iso-CBI and iso-CI analogs of CC-1065 duocarmycins

ABSTRACT

A series of bioactive analogs of (+)-CC-1065 (1) and the duocarmycins 2 and 3 are synthesized. The bioactive analogs include either iso-CI or iso-CBI (6 and 7) as a DNA alkylation subunit. Conjugated to the DNA alkylating subunits are a variety of DNA binding subunits. The bioactive analogs maintain their DNA selectivity and display enhance reactivity.

This application is a continuation of Ser. No. 09/529,345 filed Apr. 12,2000, now U.S. Pat. No. 6,262,271 which is a 371 of CopendingInternational Application No. PCT/US98/21749, filed Oct. 14, 1998 waspublished under PCT Article 21(2) in English, which claims benefit of60/061,882, filed Oct. 14, 1997.

This invention was made with government support under Contract No. CA55276 by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF INVENTION

The invention relates to antitumor antibiotics. More particularly, theinvention relates to analogs of CC-1065 and the duocarmycins having DNAalkylation and antitumor antibiotic activities.

BACKGROUND

(+)-CC-1065 (1) and the duocarmycins 2 and 3, illustrated in FIG. 1, arenatural products having antitumor antibiotic activity through thealkylation of DNA. (Hanka, L. J., et al. J. Antibiot. 1978, 31, 1211;Yasuzawa, T., et al., Chem. Pharm. Bull. 1995, 43, 378; and Takahashi,I., et al., J. Antibiot. 1991, 44, 1045.) Prior studies have shown thatthe natural products can withstand and may benefit from significantstructural modifications to the alkylation subunit and that theresulting agents retain their ability to participate in thecharacteristic sequence-selective DNA alkylation reaction. (Boger, D.L., et al., Chem. Rev. 1997, 97, 787.) These structural modifications,and the definition of their effects have served to advance theunderstanding of the origin of the catalysis of the DNA alklationreaction by 1-3. (Harper, D. E. J. Am. Chem. Soc. 1994, 116, 7573; andWarpehoski, M. A., et al., J. Am. Chem. Soc. 1995, 117, 2951.)

These structural modifications have also served to advance theunderstanding of the origin of the DNA sequence selectivity of 1-3.(Warpehoski, M. A. In Advances in DNA Sequence Specific Agents; Hurley,L. H., Ed.; JAI: Greenwich, Conn., 1992; Vol. 1, p 217; Hurley, L. H.and Draves, P. In Molecular Aspects of Anticancer Drug-DNA Interactions;Neidle, S. and Waring, M., Eds.; CRC: Ann Arbor, 1993; Vol. 1, p 89; andAristof P. A In Advance in Medicinal Chemistry, JAI: Greenwich, Conn.,1993; Vol. 2, p 67). Two models have been proposed to explain themechanism of the DNA sequence selectivity of 1-3. One model proposed byBoger states that the DNA sequence selectivity of 1-3 is determined bythe AT-rich noncovalent binding selectivity of these agents and theirsteric accessibility to the adenine N3 alkylation site. (Boger, D. L.,et al., Angew. Chem., Int. Ed. Engl. 1996, 3.5, 1439; and Boger, D. L.,et al., Biorg. Med Chem. 1997, 5, 263.) This noncovalent binding modelaccommodates and explains the reverse and offset 5 or 3.5 base-pairAT-rich adenine N3 alkylation selectivities of the natural and unnaturalenantiomers of 1 (Boger, D. L., et al., J. Am. Chem. Soc. 1990, 112,4623; and Boger, D. L., et al, Bioorg. Med Chem. 1994, 2, 115) and thenatural and unnatural enantiomers of 2-3. (Boger, D. L., et al., J. Am.Chem. Soc. 1993, 115, 9872; and Boger, D. L., et al., J. Am. Chem. Soc.1994, 116, 1635.) This noncovalent binding model also requires thatsimple derivatives of the alkylation subunits exhibit alkylationselectivities distinct from the natural products. It also offers anexplanation for the identical alkylation selectivities of bothenantiomers of such simple derivatives (5′-AA>5′-TA), and the moreextended AT-rich selectivity of the advanced analogs of 1-3 correspondsnicely to the length of the agent and the size of the required bindingregion surrounding the alkylation site. This model is further supportedby the demonstrated AT-rich noncovalent binding of these agents. (Boger,D. L., et al., Chem.-Biol. Interactions 1990, 73, 29; and Boger, D. L.,et al., J. Org. Chem. 1992, 57, 1277.) The model is also supported bythe correspondance between the observed preferential noncovalent bindingand the observed DNA alkylation of these agents. (Boger, D. L, et al.,Bioorg. Med. Chem. 1996, 4, 859.) Also the observation that thecharacteristic DNA alkylation selectivity of these agents does notrequire the presence of the C-4 carbonyl or even the activatedcyclopropane provides further support for the model (Boger, D. L. etal., J. Am. Chem. Soc. 1991, 113, 3980.; and Boger, D. L., et al. Proc.Natl. Acad. Sci. U.S.A. 1991, 88, 1431.) The accuracy of this model isfurther demonstration of the complete switch in the inherentenantiomeric DNA alkylation selectivity that accompanied the reversal ofthe orientation of the DNA binding subunits with reversed versusextended analogs of the duocarmycins. (Boger, D. L., et al., J. Am.Chem. Soc. 1997, 119, 4977; Boger, D. L., et al., J. Am. Chem. Soc.1997, 119, 4987; and Boger, D. L., et al., J. Am. Chem. Soc. 1995, 117,1443.)

The above AT-rich noncovalent binding model contrasts with analternative proposal in which a sequence-dependent backbone phosphateprotonation of the C-4 carbonyl activates the agent for DNA alkylationand controls the sequence selectivity. (Hurley, L. H. J. Am. Chem. Soc.1995, 117, 2371.)

Structural studies of DNA-agent adducts, ¹⁷⁻¹⁹ the C-4 carbonyl of thenatural products projects out of the minor groove lying on the outerface of the complexes potentially accessible to the phosphate backbone.(Lin, C. H., et al., J. Mol. Biol. 1995, 248, 162.; and Smith, J. A., etal., J. Mol. Biol. 1997, 272, 237.) However, the relative importance ofthe C-4 carbonyl positioning to the properties of these agents has notbe determined.

What is needed is a series of analogs of (+)-CC-1065 (1) and theduocarmycins 2 and 3 which exploit the AT-rich noncovalent binding modeland which retain their DNA binding and alkylating activity andselectivity. What is needed is series of analogs of (+)-CC-1065 (1) andthe duocarmycins 2 and 3 which incorporate of iso-CI and iso-CBI (6 and7). Iso-CI and iso-CBI (6 and 7) are analogs of the CI and CBIalkylation subunits 4 and 5 wherein the key C-4 carbonyl is isomericallyrelocated to the C-6 or C-8 positions, now ortho to the cyclopropane, asillustrated in FIG. 2. If the AT-rich noncovalent binding model iscorrect, the relocated carbonyls of iso-CI and iso-CBI (6 and 7) wouldproject into the minor groove inaccessible to the phosphate backbone ifparticipating in an analogous adenine N3 alkylation reaction.

SUMMARY

A series of bioactive analogs of (+)-CC-1065 (1) and the duocarmycins 2and 3 are synthesized. Each of the analogs includes iso-CI or iso-CBI (6and 7) as a DNA alkylation subunit. The novel DNA alkylation subunitsare then conjugated to known DNA binding subunits to form bioactiveanalogs of (+)-CC-1065 (1) and the duocarmycins 2 and 3. Preferred DNAbinding subunits are disclosed herein and in U.S. patent applicationSer. No. 09/051,264, incorporated herein by reference.

2-(tert-Butyloxycarbonyl)-1,2,9,9a-tetrahydrocyclo-propa[c]benzo[f]-indol-8-one(31, N-BOC-iso-CBI) and1-(tert-butyloxycarbonyl)-4-hydroxy-3-[[(methanesulfonyl)oxy]methyl]-2,3-dihydroindole(19, seco-N-BOC-iso-CI) serve as preconjugate forms to the DNAalkylating subunits, i.e., iso-CI or iso-CBI (6 and 7). The approach forsynthesizing compounds 31 and 19 was based on a directed orthometallation of an appropriately functionalized benzene (13) ornaphthalene at (24) precursor to regziospecifically install iodine atthe C-2 position. Conversion of these respective intermediates to thedihydroindole skeleton utilized an established 5-exo-trig aryl radicalcyclization onto an unactivated alkene with subsequent TEMPO trap or themore recent 5-exo-trig aryl radical cyclization onto a vinyl chloridefor direct synthesis of the immediate precursors. Closure of theactivated cyclopropane to complete the iso-CBI nucleus was accomplishedby a selective ortho spirocyclization.

Resolution and synthesis of a fill set of natural product analogs andsubsequent evaluation of their DNA alkylation properties revealed thatthe iso-CBI analogs react at comparable rates and retain the identicaland characteristic sequence selectivity of CC-1065 and the duocarmycins.This observation is inconsistent with the prior art proposal that asequence-dependent C-4 carbonyl protonation by strategically located DNAbackbone phosphates controls the DNA alkylation selectivity but isconsistent with the proposal that it is determined by the AT-richnoncovalent binding selectivity of the agents and the stericaccessibility of the N3 alklation site.

Solvolysis studies indicate that the iso-CBI-based agents have astability comparable to that of CC-1065 and duocarmycin A and a greaterreactivity than duocarmycin SA (6-7×). Solvolysis studies indicate alsoindicate that the iso-CBI-based agents are more reactive than thecorresponding CBI-based agents (5×).

Confirmation that the DNA alkylation reaction is derived from adenine N3addition to the least substituted carbon of the activated cyclopropaneand its quantitation (95%) was established by isolation andcharacterization of the depurination adenine N3 adduct. Consistent withpast studies and in spite of the deep-seated structural change in thealkylation subunit, the agents were found to exhibit potent cytotoxicactivity that correlates with their inherent reactivity.

One aspect of the invention is directed to DNA alkylating compoundshaving a DNA alkylating subunit covalently linked to a DNA bindingsubunit. The DNA alkylating compound is represented by the followingstructure:

A preferred DNA binding subunit is a radical represented by thefollowing structure:

In the above structure, A is selected from the group consisting of NHand O. B is selected from the group consisting of C and N. R₂ isselected from the group consisting of hydrogen, hydroxyl,O-alkyl(C1-C6), N-alkyl(C1-C6)₃ and a first N-substituted pyrrolidinering. R₃ is selected from the group consisting of hydrogen, hydroxyl,O-alkyl(C1-C6), N-alkyl(C1-C6)₃, the first N-substituted pyrrolidinering. R₄ is selected from the group consisting of hydrogen, hydroxyl,O-alkyl, (C1-C6), and N-alkyl(C1-C6)₃. R₅ is selected from the groupconsisting of hydrogen, hydroxyl, O-alkyl(C1-C6), and N-alkyl(C1-C6)₃ V₁represents a first vinylene group between R₂ and R₃. However, there arevarious provisos. If R₂ participates in the first N-substitutedpyrrolidine ring, then R₃ also participates in the first N-substitutedpyrrolidine ring. If R₃ participates in the first N-substitutedpyrrolidine ring, then R₂ also particlates in the first N-substitutedpyrrolidine ring. If R₂ and R₃ participate in the first N-substitutedpyrrolidine ring, then R₄ and R₅ are hydrogen. If R₂ is hydrogen, thenR₄ and R₅ are hydrogen and R₃ is N-alkyl (C1-C6)₃. The firstN-substituted pyrrolidine ring is fused to the first vinylene groupbetween R₂ and R₃ and is represented by the following structure:

In the above structure V₁ represents the first vinylene group between R₂and R₃. R₆ is selected from the group consisting of —CH₂CH₃(alkyl),—NHCH₃(—N-alkyl), —OCH₃(O-alkyl), —NH₂, —NHNH₂, —NHNHCO₂ ^(t)′Bu, and aradical represented by the following structure:

In the above structure, C is selected from the group consisting of NHand O. D is selected from the group consisting of C and N. R₇ isselected from the group consisting of hydrogen, hydroxyl,O-alkyl(C1-C6), N-alkyl(C1-C6)₃, and a second N-substituted pyrrolidinering. R₈ is selected from the group consisting of hydrogen, hydroxyl,O-alkyl(C1-C6), N-alkyl(C1-C6)₃, the second N-substituted pyrrolidinering. V₂ represents the second vinylene group between R₇ and R₈.However, the following provisos pertain. If R₇ participates in theN-substituted pyrrolidine ring, then R₈ also particlates in theN-substituted pyrrolidine ring. If R₈ participates in the N-substitutedpyrrolidine ring only if R₇ also particlates in the N-substitutedpyrrolidine ring. The second N-substituted pyrrolidine ring is fused tothe second vinylene group between R₇ and R₈ and is represented by thefollowing structure:

In the above structure, V₂ represents the second vinylene group betweenR₇ and R₈. R₉ is selected from the group consisting of —CH₂CH₃(alkyl),—NHCH₃(—N-alkyl), —OCH₃(O-alkyl), —NH₂, —NHNH₂, and —NHNHCO₂ ^(t)′Bu.

Preferred examples include DNA alkylating compounds represented by thefollowing structures:

Another aspeact of the invention is directed to DNA alkylating compoundsrepresented by the following structure:

In the above structure, R₁₃ is selected from the group consisting of—C1-C6 alkyl, —NHCH₃(—N-alkyl), —OCH₃(O-alkyl), —NH, —NHNH₂, —NHNHCO₂_(t)′Bu, and a radical represented by the following structure:

A preferred embodiment of this aspeact of the invention is representedby the following structure:

Further aspeacts of the invention are directed to chemical intermediaterepresented by the following structures:

Another aspeact of the invention is directed to DNA alkylating compoundshaving a DNA alkylating subunit covalently linked to a DNA bindingsubunit covalently linked said DNA alkylating subunit, wherein the DNAalkylating compound being represented by the following structure:

Preferred DNA binding subunit are as described above for the iso-CBIcompounds. Preferred examples of this aspect of the invention includeDNA alkylating compounds represented by the following structures:

Another aspect of the invention is directed to DNA alkylating compoundsrepresented by the following structure:

In the above structure R₁ is selected from the group consisting of—C1-C6 alkyl, —NHCH₃ (—N-alkyl), —OCH₃(O-alkyl), —NH₂, —NHNH₂, —NHNHCO₂^(t)Bu, and a radical represented by the following structure:

An example of this preferred embodiment is represented by the followingstructure:

An other aspect of the invention is directed to chemical intermediatespresented by the following structures:

DESCRIPTION OF DRAWINGS

FIG. 1 illustrates (+)-CC-1065 (1) and the duocarmycins 2 and 3, theunmodified natural products.

FIG. 2A illustsrates prior art DNA alkylation agents CI, CBI and CPI andnovel analog DNA alkylation agents iso-CI, iso-CBI and iso-CPI.

FIG. 2B illustsrate the interactions of CBI-TMI and iso-CBI-TMI with theminor groove of DNA.

FIG. 3A illustrates data from a solvolysis study (UV spectra) ofN-BOC-iso-CBI (31) in 50% CH₃OH-aqueous buffer (pH 3.0, 4:1:20) (v/v/v)0.1 M citric acid, 0.2 M NaH₂PO₄, and H₂O. The spectra were recorded at0, 10, 20, 28, 56, and 84 hours.

FIG. 3B illustrates data from a solvolysis study (UV spectra) of;N—CO₂Me-iso-CBI (33, middle) in 50% CH₃OH-aqueous buffer (pH 3.0,4:1:20) (v/v/v) 0.1 M citric acid, 0.2 M NaH₂PO₄, and H₂O. The spectrawere recorded at 0, 10, 21, 29, 57, and 86 hours.

FIG. 3C illustrates data from a solvolysis study (UV spectra) ofcompound 35 in 50% CH₃OH-aqueous buffer (pH 3.0, 4:1:20) (v/v/v) 0.1 Mcitric acid, 0.2 M NaH₂PO₄, and H₂O. The spectra were recorded at 0, 1,3, 5, 10, and 16 hours

FIGS. 4A-4B illustrate stick models of the side view and 90° rotationview of the activated cyclopropane of N—CO₂Me-iso-CBI and N—CO₂Me-CBI,with data taken from the X-ray crystal structures and highlighting thestereoelectronic and geometric alignment of the cyclopropane with thecyclohexadienone π-system.

FIG. 5 illustrates thermally-induced strand cleavage of w794 DNA (SV40DNA segment, 144 bp, nucleotide nos. 138-5238); DNA-agent incubation for24 hours at 25° C., removal of unbound agent and 30 minutes ofthermolysis (100° C.), followed by denaturing 8% PAGE andautoradiography, lane 1, control DNA; lanes 2-5, Sanger G, C, A, and Tsequencing reactions; lanes 6-7 (−)-iso-CBI-TMI (1×10⁻³ and 1×10⁻⁴ M);lanes 8-9, (+)-CBI-TMI (1×10⁻⁵ and 1×10⁻⁴ M); lanes 10-11,(+)-duocarmycin SA (1×10⁻⁵ and 1×10⁻⁶).

FIG. 6 illustsrates thermally-induced strand cleavage of w836 DNA (146bp, nucleotide nos. 5189-91): DNA-agent incubation for 24 h((−)-iso-CBI-TMI) at 25° C., removal of unbound agent and 30 min ofthermolysis (100° C.), followed by denaturing 8% PAGE andautoradiography; lane 1, control DNA; lanes 2-5, Sanger G, C, A, and Tsequencing reactions; lanes 6-8 (−)-iso-CBI-TMI (1×10³ to 1×10⁻⁵ M);lanes 9-10, (+)-duocarmycin SA (1×10⁻⁵ to 1×10⁻⁶ M); lanes 11-12,(+)-iso-CBI-TMI (1×10⁻² and 1×10⁻³).

FIG. 7 illustrates thermally-induced strand cleavage of w794 DNA (SV40DNA segment, 144 bp, nucleotide nos. 138-5238); DNA-agent incubation for24 h at 25° C., removal of unbound agent and 30 min of thermolysis (100°C.), followed by denaturing 8% PAGE and autoradiography; lane 1, controlDNA; lanes 2-5, Sanger G, C, A, and T sequencing reactions; lanes 6-8(+)-duocarmycin SA (1×10⁻⁴ to 1×10⁻⁶ M); lanes9-11, seco-iso-CI-TMI(1×10⁻³ to 1×10⁻⁵ M).

FIGS. 8A-8E illustrate IC₅₀ values for the indicated families of DNAalkylating agents.

FIGS. 9A-9C illustrate solvolysis data for the indicated compounds.

FIG. 10 provides ¹H and ¹³C NMR data of various adenine adducts.

DETAILED DESCRIPTION

Sundberg and co-workers report the first synthesis of agents isomeric tothe natural products. (Sundberg, R. J., et al., Tetraedron Lett. 1983,24, 4773; and Sundberg, R. J., et al., J. Org. Chem., 1991, 56, 3048.)An agent isomeric with the alkylation subunit of CC-1065 was preparedemploying an intramolecular

carbene insertion of an o-quinonediazide onto a tethered alkene.Presumably because of the perceived unique character of the authenticalkylation subunit at the time of the work, its chemical behavior,biological characteristics, and DNA alkylation properties were notexamined.

As illustrated above in Scheme I, an alternative route was subsequentlydevised for the synthesis of the isomeric CI analogs (Boger, D. L., etal., J. Am. Chem. Soc. 1990, 112, 5230.) and for CBI analogs. (Boger, D.L., et al., J. Org. Chem. 1995, 60, 1271; Boger, D. L. et al., J. Org.Chem. 1992, 57, 2873; Boger, D. L., et al., J. Am. Chem. Soc. 1989, 111,6461; Boger, D. L., et al., J. Org. Chem. 1990, 55, 5823; Boger, D. L.,et al., Tetrahedron Lett. 1990, 31, 793; Boger, D. L., et al., Bioorg.Med. Chem. Lett. 1991, 1, 55; Boger, D. L., et al., Bioorg. Med. Chem.Lett. 1991, 1, 115; Boger, D. L., et al., J. Am. Chem. Soc. 1992, 114,5487; and Boger, D. L., et al., Bioorg. Med. Chem. 1995, 3, 611.) Thestrategy is complementary to our synthesis of CBI in which thedihydroindole skeleton was constructed by a 5-exo-trig radicalcyclization of an aryl radical onto a tethered alkene and its extensionto the isomeric analogs required C2 versus C4 regiocontrol in the keyaromatic halogenation step. In the synthesis of CI and CBI, aregioselective electrophilic halogenation served to install iodine orbromine para to the phenol ether. For the isomeric agents, an orthohalogenation protocol was required and a cooperative directed orthometallation²³ was implemented to install the C2 halide. (Snieckus, V.Chem. Rev. 1990, 90, 879.) Its adoption required two easily removedcooperative directing metallation groups: OMOM (Winkle, M. R, et al., J.Org. Chem. 1982, 47, 2101) and NHBOC. (Muchkowski, J. M., et al., J.Org. Chem. 1980, 45, 4798.) In addition, the Winstein para Ar-3′spirocyclizaton (Baird, R., et al., J. Am. Chem. Soc. 1963, 85,567;1962, 84, 788; 1957, 79, 756.) utilized to close the cyclopropane inthe CI and CBI synthesis, is now replaced by an ortho spirocyclizationrequiring C-alkylation with cyclopropane formation (Brown, R. F. C., etal., Tetrahedron Lett. 1981, 22, 2915; Smith III A. B., et al.,Tetrahedron Lett. 1987, 28, 3659; and Kigoshi, H, et al., TetrahedronLett. 1997, 38, 3235) rather than competitive O-alkylation anddihydrofuran formation.

This approach was first examined with iso-CI employing the commerciallyavailable 3-nitrophenol (10), Scheme 2. Protection of the phenol as theMOM ether 11 (NaH, MOMCl, Bu₄NI 89%), reduction of the nitro group with

Al—Hg amalgam (Et₂O—H₂O, 89%) (Meyers, A. I., et al., J. Org. Chem.1975, 40, 2021.), and BOC protection of the free amine 12 (BOC₂O, >95%)provided 13, a key intermediate with which to examine the directed orthometallation Treatment of 13 with 3.5 equiv of n-BuLi and TMEDA at −20°C. (2 h) in THF, and reaction of the the aryl lithium intermediate with1-chloro-2-iodoethane provided 14 (46%) along with recovered startingmaterial (41%). Although not extensively examined, this conversion wasnot improved through use of longer reaction times, different reactiontemperatures or solvents, or additional amounts of n-BuLi. However, theconversion did allow the synthesis to proceed and, as detailed, thisreaction proved much more effective in the iso-CBI series where it wasmore carefully optimized. N-Alkylation with allyl bromide (NaH, >95%)was followed by Bu₃SnH promoted 5-exo-trig free radical cyclization of15 with in situ TEMPO trap of the resulting primary radical to provide16 (91%). Subsequent reduction of the N—O bond (Zn, HOAc, 87%) withoutthe competitive deprotection of either the MOM ether or the N-BOCprotecting groups afforded the free alcohol 17 in excellent overallyield. Activation of the primary alcohol (MsCl, Et₃N, 94%) afforded thekey intermediate 18, further detailed below.

In order to prepare N-BOC-iso-CI for direct comparison with prioragents, selective removal of the MOM group in the presence of the BOCgroup was required. Mild acid-catalyzed deprotection (HCl, i-PrOH/THF,48%) provided seco-N-BOC-iso-CI (19) accompanied by 41% recovery ofstarting material. Although not optimized, this selective deprotectionfound greater success in the synthesis of N-BOC-iso-CBI where it wasmore closely examined. Exhaustive deprotection of the MOM ether and theBOC group (3.6 M HCl/EtOAc) followed by EDCI-promoted coupling with5,6,7-trimethoxyindole-2-carboxylic acid (TMI-COOH)^(10.31) provided 20(55% overall). (EDCI=1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride; Boger, D. L., et al., J. Org. Chem. 1990, 55, 4499.)Consistent with expectations, spirocyclization of 19 to N-BOC-iso-CI(21)

could be effected by treatment with DBU, but its exceptional reactivityprecluded attempts to isolate and characterize the agent (eq1).(Diagnostic ¹H NMR (CD₃CN, 400 MHz) signals for 21 generated in situ:δ 2.79 (dt, J=5.4, 7.8 Hz, 1H), 1.75 (dd, J=3.1, 7.8 Hz, 1H).)Sufficient for our considerations, the studies revealed thatN-BOC-iso-CI is more reactive than its counterpart N-BOC-CI which,

although exceptionally reactive, can be isolated by quick columnchromatography. Since the seco agents can be expected to behaveanalogous to their ring-closed cyclopropane counterparts in biologicalassays and DNA alkylation studies, and given that the carefulcomparisons could be made with the more stable iso-CBI based agents,their isolation and characterization were not further pursued.

With the additional stability provided by the fused benzene ring, theCBI analogs are substantially more stable and biologically more potentthan the CI series. The extension of this approach to the preparation ofof the iso-CBI alkylation subunit is detailed in Scheme 3 and providedthe opportunity to accurately document the effects of this deep-seatedstructural change. Starting with 22, available in two steps (70%) fromcommercially available 1,3-dihydroxynaphthalene, protection of thephenol (MOMCl, NaH, Bu₄NI, 78%) as the MOM ether provided the directedortho metallation substrate 23. Treatment of 23 with 3.5 equiv of n-BuLiand TMEDA at −25° C. in THF for 2 h and reaction of the aryl lithiumintermediate with 1-chloro-2-iodoethane gave the C2 iodide 24 in 80%yield, a substantial improvement over the iso-CI directed metallation.N-Alkylation (NaH, allyl bromide, 94%), 5-exo-trig free radicalcyclization of 25 with in situ TEMPO trap (Bu₃SnH, TEMPO, 94%), and N—Obond reduction of 26 (Zn, HOAc, 87%) provided the primary alcohol 27 inexcellent overall yield. Conversion to the chloride 28 upon activationof the primary alcohol under Mitsunobu conditions (Ph₃P, CCl₄, 90%)provided a resolvable intermediate that served as a penultimateprecursor to all analogs. A 5-exo-trig radical cyclization onto atethered vinyl chloride was shown to provide the 5-membered ring with asuitable leaving group already in place. This concise strategy wasadopted for the synthesis of iso-CBI (eq 2). Thus, N-alkylation of 25with 1,3-dichloropropene proceeded in 96% yield providing the keyradical cyclization precursor (29). Treatment with catalytic AIBN (0.1equiv) and Bu₃SnH (1.1 equiv) at 80° C. (C₆H₆) yielded the tricycliccore of iso-CBI (28) in 96% yield. This improved approach shortens theoriginal synthesis by two steps, avoiding reductive removal of the TEMPOgroup and conversion to 28. With this improvement, the preparation of 28requires 4 steps and proceeds in 58% yield overall. Removal of the MOMether (HCl, i-PrOH/THF, 90%) without competitive N-BOC deprotectionafforded seco-N-BOC-iso-CBI (30) in superb yield. Ortho spirocyclization(DBU, CH₃CN, 96%) provided N-BOC-iso-CBI (31) which could be purified bystandard chromatography. Similarly, exhaustive deprotection of 28 (3.6 NHCl/EtOAc) followed by N-acylation with methyl chloroformate provided 32(65%) and spirocycization (DBU, CH₃CN, 25° C., 91%) afforded 33.

Initial attempts to synthesize iso-CBI (34) by exhaustive deprotection(3.6 M HCl/EtOAc) and subsequent ring closure (5% aqueous NAHCO₃/THF)conducted in the presence of air resulted in the isolation of thequinone 35 (Scheme 4). Presumably, adventious oxidation of theintermediate iso-CBI subsequent to spirocyclization provided 35 and aninteresting further modification of the iso-CBI alkylation subunit.(Such agents may be subject to reductive activation.) Consistent

with this, spirocyclization conducted in an aprotic solvent under aninert atmosphere (2.2 equiv DBU, CH₃CN, 25° C., 20 min, Ar) provided theunstable iso-CBI (34, 55%) and subsequent exposure of 34 to air resultedin conversion to 35.

Exclusive spirocyclization with cyclopropane formation was observed, andno competitive O-alkylation leading to 36 was detected. However,prolonged exposure of neat 31 to ambient light at 25° C. (48 h) did leadto carbonylcyclopropane rearrangement to form 36 (eq 3). (Wong, H. N.C., et al., Chem. Rev. 1989, 89, 165.) An identical neat sample of 31protected from light by foil remained unchanged after 48 h. Thus, thehandling and storage of the iso-CBI based agents were conductedminimizing their exposure to light typically at subzero temperatures.Examination of the compounds over time showed no appreciabledecomposition or rearrangement under these storage conditions.

Resolution: In order to assess the properties of both enantiomers of theiso-CBI based agents, a direct chromatographic resolution of 28 on asemipreparative ChiralCel OD column (2×25 cm, 3% i-PrOH/hexane, α=1.24)was utilized. (Boger, D. L., et al., J. Am. Chem. Soc. 1994, 116, 7996.)This procedure provided both enantiomers (>99% ee) of an advancedintermediate and avoided diastereomeric derivatization, separation, anddederivatization. The assignment of absolute configuration was based onthe conversion of the slower eluting enantiomer of 28 (t_(R)=35.8 min)to 32 and subsequent single-crystal X-ray structure determination whichrevealed the unnatural (3R)-configuration (Scheme 3). (The atomiccoordinates for this structure have been deposited with the CambridgeCrystallographic Data Centre and may be obtained upon request from theDirector, Cambridge Crystallographic Data Centre, 12 Union Road,Cambridge, CB2 1EZ, UK) Consistent with this assignment, the agentsderived from the (3S)-enantiomer analogous to the absolutestereochemistry found in the natural products 1-3 exhibited the morepotent biological activity, the more effective DNA alkylationproperties, and exhibited a DNA alkylation selectivity identical to thenatural products.

Synthesis of Duocarmycin and CC-1065 Analogs: The iso-CBI alkylationsubunit was incorporated into CC-1065 and duocarmycin analogs asdetailed in Scheme 5. Exhaustive deprotection of 28 (3.6 M HCl/EtOAc, 30min) followed by immediate coupling (3 equiv of EDCI, DMF, 25° C.) ofthe amine hydrochloride salt with 5,6,7-trimethoxyindole-2-carboxylicacid (37, 3 h, 91%), 38 (3 h, 95%), 39 (3 h, 87%), indole₂ (40, 3 h,74%), CDPI₁ (41, 9 h, 80%), and CDPI₂ (42, 12 h, 32%) provided 43, 45,47, 49, 51 and 53, respectively. (Boger, D. L., et al., Bioorg. Med.Chem. 1995, 3, 1429; Boger, D. L., et al., J. Org. Chem. 1987, 52, 1521;and Boger, D. L., et al., J. Org. Chem. 1984, 49, 2240.) The poorsolubility of CDPI₂ precluded efficient coupling and isolation,resulting in a lower yield. DBU (1.5 equiv, 25° C.) spirocyclization of43 (CH₃CN, 30 min, 94%), 45 (CH₃CN, 30 min, 85%), 47 (CH₃CN, 30 min,83%), 49 (DMF, 30 min, 88%), 51 (DMF, 60 min. 81%), and 53 (DMF, 60 min,59%) afforded 44, 46, 48, 50, 52 and 54, respectively, in excellentconversions.

Solvolysis Reactivity and Regioselectivity: Two fundamentalcharacteristics of the alkylation subunits have proven important in paststudies. (Boger, D. L., et al., Angew. Chem., Int. Ed. Engl. 1996, 35,1439.) The first is the stereoelectronically-controlled acid-catalyzedring opening of the activated cyclopropane which dictates preferentialaddition of a nucleophile to the least substituted cyclopropane carbon.The second is the relative rate of acid-catalyzed solvolysis which hasbeen found to accurately reflect the functional reactivity of the agentsand to follow a direct relationship between solvolysis stability and invitro cytotoxicity. While N-BOC-iso-CBI retains many of the structuralcharacteristics of N-BOC-CBI, the isomeric modifications which maydisrupt the vinylogous amide conjugation were anticipated to reduce thesolvolytic stability of the agents. However, it was not clear what themagnitude of this effect might be nor whether solvolysis would stilloccur with the same high regioselectivity.

N-BOC-iso-CBI(31, t_(1/2)=27.6 h, k=6.97×10⁻⁶ s⁻¹) and N—CO₂Me-iso-CBI(33, t_(1/2)=30.1 h, k=6.40×10⁻⁶ s⁻¹) proved to be reasonably stabletoward chemical solvolysis at pH 3 exhibiting a reactivity comparable tothe CC-1065 alkylation subunit (N-BOC-CPI, 55, t_(1/2)=36.7 h), but morestable than the duocarmycin A alkylation subunit (N-BOC-DA, 56,t_(1/2)=11 h), Table 1. However,

TABLE 1 Solvolysis Reactivity and Regioselectivity

k t_(1/2) Agent (s⁻¹, pH 3) (h, pH 3) Regioselectivity 31 6.98 × 10⁻⁶ 28h 40:1  35 3.80 × 10⁻⁵ 5 h nd 55 5.26 × 10⁻⁶ 37 h 4:1 56 1.75 × 10⁻⁵ 11h 3:2 57 1.08 × 10⁻⁶ 177 h 6-4:1   58 1.45 × 10⁻⁶ 133 h >20:1  59 1.75 ×10⁻⁶ 110 h >20:1  60 0.99 × 10⁻⁶ 194 h >20:1  61 1.98 × 10⁻² 0.01 h nd

N-BOC-iso-CBI was significantly less stable than N-BOC-DSA (57,t_(1/2)=177 h) and N-BOC-CBI (58, t_(1/2)=133 h). Thus, N-BOC-iso-CBI(31) proved to be 5× more reactive than its direct comparison analogN-BOC-CBI. The solvolysis was followed spectrophotometrically by UV withthe disappearance of the long-wavelength absorption band of the iso-CBIchromophore (397 nm), FIG. 3. The reactivity of the quinone 35 was alsoexamined at pH 3 (t_(1/2)=5.1 h, k=3.80×10⁻⁵ s⁻¹) and it proved to be5-6× more reactive than 31 or 33.

The acid-catalyzed nucleophilic addition of CH₃OH to 31 was conducted ona preparative scale to establish the regioselectivity of addition, andconfirmed by synthesis of the expected product 62 derived fromnucleophilic addition to the least substituted cyclopropane carbon.Treatment of N-BOC-iso-CBI with 0.1 equiv of CF₃SO₃H in CH₃OH (25° C.,17 h) resulted in the clean solvolysis (94%) to provide a 40:1 mixtureof 62 and 63 (Scheme 6). Consequently, the acid-catalyzed CH₃OH additionto 31 occurs with near exclusive regioselectivity (40:1) analogous toN-BOC-CBI (58, >20:1)²² which is much more selective than the naturalalkylation subunits themselves (6-1.5:1). (Boger, D. L., et al., Bioorg.Med. Chem. Lett. 1996, 16, 1955; Boger, D. L., et al., J. Am. Chem. Soc.1997, 119, 311.)

X-Ray Structure of N—CO₂Me-iso-CBI (33): Structural Correlation withSolvolysis Regioselectivity and Reactivity. The single-crystal X-raystructure determination of N—CO₂Me-iso-CBI (33) was conducted and bydirect comparison with that of N—CO₂Me-CBI⁴² established a structuralbasis for the observed properties (FIG. 4). (Boger, D. L.; Turnbull, P.J. Org. Chem. 1997, 62, 0000.) The first striking similarity between thetwo systems is the perpendicular orientation of the bent orbital of thecyclopropane bond extending to the least substituted C9 cyclopropanecarbon. This idealized stereoelectronic alignment with the developingπ-system of the solvolysis product phenol imposes a preference fornucleophilic addition to the less substituted C9 cyclopropane carbon. Incontrast, the cyclopropane bond extending to the tertiary C9 a carbon isnearly orthogonal to the π-system of the cyclohexadienone, and S_(N)2addition to this carbon is disfavored stereoelectronically as well assterically. The relative cyclopropane bond lengths of iso-CBI reflectthis orientation and n conjugation in which the breaking C9-C8acyclopropane bond (1.531 Å) is longer, and thus weaker, than the C9a—C8abond (1.513 Å) extending to the more substituted carbon. Although thering expansion solvolysis would place a developing positive charge on apreferred secondary versus primary carbon, this inherent preference isoverridden by the stereoelectronic control of the reactionregioselectivity as well as the characteristics of a S_(N)2 reactionwhich prefer attack at the less substituted center.

In addition, the X-ray structures have provided insights into the originof the difference in stability between CBI and iso-CBI. The stability ofCC-1065, the duocarmycins and their analogs is a result of at leastthree structural features: the conjugative stability provided by thefused aromatic system, the non-ideal alignment of the cyclopropane, andthe strong cross-conjugative stability provided by the vinylogous amide.The first of these features, the fused aromatic ring, is present in bothiso-CBI and CBI. Like the CI/CBI comparison, the iso-CI/iso-CBIreactivity comparison reveals that the diminished aromatization drivingforce for iso-CBI relative to iso-CI contributes significantly to thestability. In addition, The cyclopropane alignments with the π-systemsin N—CO₂Me-iso-CBI and N—CO₂Me-CBI are similar. Both are bisected by theplane of the cyclohexadienone nearly equally (41°/15° for iso-CBI and41°/16° for CBI). In both, the cyclopropane is not only pulled down butalso to the side by the constraints of the fused 5-membered ring (9° foriso-CBI and 12° for CBI). Thus, both benefit in stability from thenon-ideal alignment and conjugation of the cyclopropane which is imposedby the fused 5-membered ring. The important distinction between the twosystems is the direct cross-conjugated stability afforded the activatedcyclopropane by the vinylogous amide. Diagnostic of this vinylogousamide conjugation is the shortened length of the N²—C^(2a) bondreflecting this resonance stabilization. As this cross-conjugatedvinylogous amide stabilization decreases, the conjugation and inherentreactivity of the cyclopropane correspondingly increases. Consistentwith this, N—CO₂Me-iso-CBI exhibits a N²—C^(2a) bond length (1.400 Åversus 1.426 Å for 32) indicative of a diminished but not eliminatedvinylogous amide conjugation relative to N—CO₂Me-CBI (1.390 Å versus1.416 Å for seco N—CO₂Me-CBI)⁴² and that follows trends established inrecent studies (Table 2).

TABLE 2 agent N²—C^(2a) bond length (Å) t_(½) (h, pH 3) N—CO₂Me-CBI⁴²1.390 133 N—CO₂Me-iso-CBI 1.400 28 N—BOC—CBQ⁴³ 1.415 2.1 N—CO₂Me—CNA⁴²1.428 0.03

DNA Alkylation Selectivity and Efficiency: The DNA alkylation propertiesof the agents were examined within w794 and w836 duplex DNA for whichcomparative results are available for related agents. (Boger, D. L., etal., J. Am. Chem. Soc. 1994, 116, 11335; Boger, D. L., et al., J. Am.Chem. Soc. 1994, 116, 6461; and Boger, D. L., et al., J. Am. Chem. Soc.1995, 117, 11647) The alkylation site identification and the assessmentof the relative selectivity among the available sites were obtained bythermally-induced strand cleavage of the singly 5′ end-labeled duplexDNA after exposure to the agents. Following treatment of the end-labeledduplex DNA with a range of agent concentrations and temperatures in thedark, the unbound agent was removed by EtOH precipitation of the DNARedissolution of the DNA in aqueous buffer, thermolysis (100° C., 30min) to induce strand cleavage at the sites of DNA alkylation,denaturing high-resolution polyacrylamide gel electrophoresis (PAGE)adjacent to Sanger dideoxynucleotide sequencing standards, andautoradiography led to identification of the DNA cleavage and alkylationsites. The full details of this procedure have been disclosed elsewhere.(Boger, D. L., et al., Tetrahedron 1991, 47, 2661.)

A representative comparison of the DNA alkylation by (−)-iso-CBI-TMI(44) alongside that of (+)-duocarmycin SA and (+)-CBI-TMI is illustratedin FIG. 5. There are three important conclusions that can be drawn fromthese comparisons. First, (−)-iso-CBI-TMI alkylates DNA in a manneridentical to (+)-duocarmycin duocarmycin SA and (+)-CBI-TMI exhibitingthe same sequence selectivity. No new sites of alkylation were detected,and only adenine N3 alkylation was detected under the conditions oflimiting agent and excess DNA Notably, such sequencing studies onlydetect the higher affinity alkylation sites and minor sites ofcomparable affinities (1-0.01×). Under these conditions, the studiesillustrate that iso-CBI-TMI, like duocarmycin SA and CBI-TMI, exhibitsan exclusive preference for adenine versus guanine N3 alkylation.However, given the reactivity of iso-CBI-TMI, it is likely that a minorguanine alkylation could be expected at incubations carried out athigher agent-base pair ratios analogous to that observed with the morereactive agents including CC-1065 (Park, H.-J., et al., J. Am. Chem.Soc. 1997, 119, 629.) and duocarmycin A.⁴⁶ (Sugiyama, H., et al.,Tetrahedron Lett. 1993, 34, 2179; Yamamoto, K, et al., Biochemistry1993, 32, 1059; and Asai, A, et al., J. Am. Chem. Soc. 1994, 116, 4171.)Importantly, the identical behavior of (−)-iso-CBI-TMI and (+)-CBI-TMIillustrate that the position of the C-4 carbonyl does not influence thesequence selectivity of the DNA alkylation. This is inconsistent withthe proposal of a sequence-dependent phosphate backbone protonation ofthe C-4 carbonyl for activation of the agent for DNA alkylation thatcontrols the sequence selectivity. It is, however, fully consistent withthe model in which it is controlled by the AT-rich noncovalent bindingselectivity of the agents and their steric accessibility to the adenineN3 alkylation sites.

Secondly, although there are no distinctions in the sequenceselectivity, there is a significant difference in the relativeefficiencies of DNA alkylation. Consistent with its relative reactivityand cytotoxic potency, (−)-iso-CBI-TMI alkylated DNA 50-100× lessefficiently than (+)-CBI-TMI and (+)-duocarmycin SA Thus,(−)-iso-CBI-TMI was found to alkylate DNA with an efficiency comparableto duocarmycin A which has been shown to be ca. 10× less efficient thanduocarmycin SA.^(10.11) In addition and analogous to the observationsmade with the agents containing the more reactive alkylation subunitsincluding duocarmycin A, but unlike the more stable agents, thealkylation efficiency of (−)-iso-CBI-TMI was found to increase as theincubation temperature was decreased from 25° C. to 4° C. This suggeststhat the differences may be attributed in part to the nonproductivesolvolysis of iso-CBI-TMI which competes with alkylation and lowers theoverall efficiency of DNA alkylation.

Thirdly, although the rate of DNA alkylation by 44 was not accuratelyquantitated, it is qualitatively similar to those of CBI-TMI andduocarmycin SA and much faster than agents we have examined whichexhibited substantially diminished rates (e.g. reversed analogs ofduocarmycin SA). Thus, the relocation of the C-4 carbonyl did not impactthe rate of DNA alkylation in a manner that would be consistent with aphosphate backbone protonation (or cation Lewis acid complexation)required of catalysis in the alkylation site model.

Similar results were obtained within w836 DNA (FIG. 6). The naturalenantiomer (−)-iso-CBI-TMI alkylated the same sites as (+)-duocarmycinSA but did so with a 50-100 fold lower efficiency. Within the segmentillustrated, the two natural enantiomers alkylated the first three 3′adenines in the sequence 5′-AAAAAA and less effectively the fourth 3′adenine corresponding to alkylation and 3′-5′ binding across a 3-4base-pair AT-rich site (i.e., 5′-AAAA>5′-CAAA). Like the unnaturalenenatiomer of CBI-TMI, the unnatural enantiomer, (+)-iso-CBI-TMI,alkylated DNA 50-100× less effectively than the natural enantiomer anddid so with a selectivity identical to that of ent-(-duocarmycin SA.Within w836, this constitutes the central adenines within the sequence5′-AAAAAA and corresponds to alkylation of 3-4 base-pair AT-rich siteswith binding in the reverse 5′-3′ direction. Because of thediastereomeric nature of the adducts, the unnatural enantiomer alkylatesthe second 5′ base (adenine) within the sequence (i.e.,5-AAAA>5′-CAAAA). This has been discussed in detail and illustratedelsewhere⁴ and both enantiomers of iso-CBI-TMI conform nicely to themodels. Thus, the relocation of the C-4 carbonyl from the outer face ofa bound complex potentially proximal to the phosphate backbone to aposition deep in the minor groove inaccessible to the phosphates had noimpact on the DNA alkylation selectivity of either enantiomer.

In addition, the DNA alkylation properties of 20, seco iso-CI-TMI, wereexamined alongside (+)-duocarmycin SA and a representative comparison isillustrated in FIG. 7 with w794 DNA In past studies, such seco agentshave behaved in a manner indistinguishable from their cyclopropanering-closed counterparts exhibiting identical DNA alkylationselectivities, efficiencies, and cytotoxic activity indicating that ringclosure is not limiting under the assay conditions. Since we were notable to isolate the ring closed iso-CI agents because of theirexceptional reactivity, the examination was conducted with the secoprecursor 20. Analogous to the observations made with iso-CBI-TMI, 44alkylated DNA in a manner identical to duocarmycin SA alkylating onlyadenine and exhibiting the same sequence selectivity. Although no newsites of alkylation were detected, the relative selectivity among theavailable sites was slightly lower with 20. This is illustrated nicelyin FIG. 7 where 20 alkylates the minor sites more prominently than does(+)-duocarmycin SA Interestingly and importantly, 20 alkylated DNA only10× less efficiently than (+)-duocarmycin SA being far more effectivethan the ring closed iso-CI-TMI might be projected to be. To date, thisobservation is unique to the iso-CI series and, as yet, has not beenobserved in the iso-CBI series or with other analogs incorporatingmodified alkylation subunits. Whether this stems from the use of themesylate versus chloro seco precursor for 20 or is unique to the iso-CIseries remains to be established. However, it does suggest that inselected instances the seco precursors may exhibit productive propertiesthat exceed those of the corresponding cyclopropane ring-closedmaterials especially with the more reactive agents. Unlike the morestable agents which readily undergo cyclopropane ring closure, the ringclosure of 20 to iso-CI-TMI under the conditions of the assay isunlikely. Rather, the DNA alkylation most likely occurs directly with 20without the intermediate generation of the free cyclopropane agent.Thus, not only did the relocation of the C-4 carbonyl not alter the DNAalkylation selectivity, but its removal altogether may be possibleproviding a class of agents which, depending on the nature of theelectrophile, also exhibit comparable DNA alkylation selectivities,efficiencies, and rates. This is consistent with early observations thatrelated electrophiles that lack the capabilities for ring closure to anactivated cyclopropane exhibit DNA alkylation selectivities identical tothe corresponding natural products.

TABLE 3 Calf Thymus DNA Alkylation base-pair conditions equiv 66 65 44solvolysis 4° C., 72 h 75^(a) 94% <5% nd nd 4° C., 72 h 150^(a) 95% <5%nd nd 4° C., 72 h 150^(b) 92% <5% nd nd 4° C., 120 h 150^(b) 95% <5% ndnd ^(a)Analytical scale, HPLC separation and UV quantitation.^(b)Preparative scale, isolation and weight quantitation. ^(c)nd = notdetected.

Quantitation, Isolation, and Characterization of the (-iso-CBI-TMIAdenine Adduct: The initial alkylation studies established that(−)-iso-CBI-TMI alkylated adenine within the minor groove in a manneridentical to (+)-duocarmycin SA The thermal cleavage of DNA used toidentify the alkylation sites in these studies only detects adductssusceptible to thermal glycosidic bond cleavage (adenine N3, guanine N3,or guanine N7 alkylation), and potential alkylation events involvingother nucleophilic centers in DNA may not be detected in this assay. Inorder to confirm that (−)-iso-CBI-TMI alkylates DNA in a manneridentical to (+)-duocarmycin SA and in efforts that established therelative extent of adenine N3 versus alternative alkylation events, thequantitation of the adenine N3 alkylation reaction and confirmation ofthe structure of the product of the reaction were established throughisolation and characterization of the thermally released adenine adduct.

This was addressed through a study of the alkylation of calf thymus DNA.Optimized conditions for the alkylation were established for (−)-44 onan analytical scale (100 μg of agent). For this purpose, thelong-wavelength UV absorption of the agent and the adduct provided auseful quantitative measure of the adenine adduct, unreacted startingmaterial and any side products (solvolysis or rearrangement), Table 3.Analytical HPLC analysis was used to confirm the identity of theproducts through correlation of retention times and UV spectral datawith authentic materials. The preparative DNA alkylation reaction andsubsequent isolation of (+)-66 was carried out under conditionsdetermined to provide complete consumption of the agent in the presenceof a large excess of DNA Thus, extraction (EtOAc) of the aqueous buffersolution containing calf thymus DNA following alkylation (4° C., 72 h,150 bp) afforded no recovered (−)-44, and only a small amount (<5%) ofthe rearranged product 65. Conducting the reaction at 4° C. in thepresence of a large excess of DNA precluded competitive solvolysis.Thermal treatment of the alkylated DNA in aqueous 10 mM sodium phosphatebuffer (100° C., 30 min, pH 7.0) followed by EtOAc extraction provided66 in 90-95% conversion, ≧95% purity (by HPLC). Repeating this thermaltreatment provided little or no additional adduct. No trace of acompeting guanine adduct could be detected. This high conversion to asingle adduct established that 44 participates exclusively in theadenine N3 alkylation reaction under the conditions examined.

Full characterization of 66 unambiguously established its structure andthe spectral characteristics showed strong homology to the duocarmycinSA¹¹ and A adenine N3 adducts and N3 methyl adenine (FIG. 10). In the ¹HNMR, the C3-H of adduct 66 was observed as a single proton (1H) at acharacteristic chemical shift of 4.36-4.37. The C3-H NMR signal for thebenzylic center resulting from alternative adenine N3 addition to themore substituted cyclopropane carbon would be readily distinguishableappearing as two protons (3.5-3.6 ppm, 2H) with a large geminal couplingconstant (19.5 Hz). Additionally diagnostic of the structure were thechemical shifts and coupling constants for C2-H₂, and C4-H₂.Characteristic of an adenine N3 alkylation product, the adenine C2-H andC8-H were readily distinguishable. The ¹H NMR of the protonated base wastaken in DMSO/1% TFA and again a strong correlation to the spectral dataobtained for the duocarmycin SA adduct was observed. The two adenineC6-NH₂ protons were seen as separate signals indicating restrictedrotation about the ⁶C=NH₂₊ bond as is present in protonated N-methyladenine. In addition, a downfield shift as a result of protonation ofboth the adenine C8-H and adenine C2-H was also observed. The ¹³C NMR of66 was also found to be in excellent agreement with that of theduocarmycin SA and A adducts. (Boger, D. L., et al., J. Am. Chem. Soc.1991, 113, 6645.) The key distinguishing signals are found within orproximal to the fused five- versus six-membered ring with 66 exhibitingchemical shifts consistent only with the former, i.e. C3 at δ 39.7consistent with duocarmycin SA (δ 41.1) and inconsistent with thesix-membered ring in duocarmycin B₁/C₁ (δ 33-34). Thus, the adenine N3addition to the least substituted cyclopropane carbon of(−)-iso-CBI-TMI, as with (+)-duocarmycin SA, was found to account for90-100% of the consumption of the agent in the presence of duplex DNAand confirmed that it binds and alkylates DNA in a manner identical tothe natural products despite the relocation of the C-4 carbonyl.

In Vitro Cytotoxic Activity Past studies with agents in this class havedefined a direct correlation between solvolysis stability and cytotoxicpotency. Consistent with their relative reactivity, the iso-CBI basedagents exhibited cytotoxic activity that closely followed thisrelationship in spite of the deep-seated structural modification (Table5, FIG. 8). The results, which also follow trends established in the DNAalkylation studies, demonstrate that the (−)-enantiomer of the analogspossessing the (S)-configuration analogous to the natural products, isthe more potent enantiomer by 10-50×. The exception to thisgeneralization is N-BOC-iso-CBI where the two enantiomers were notreadily distinguishable and the unnatural enantiomer was consistentlyslightly more potent (1-2×). The seco precursors, which lack thepreformed cyclopropane but possess the capabilities of ring closure,were found to possess cytotoxic activity that was indistinguishable fromthe final ring-closed agents. Consistent with the unique importance ofthe C5 methoxy group of the duocarmycins, iso-CBI-TMI (44) and 46 werefound to be equipotent illustrating that the C6 and C7 methoxy groups of44 are not contributing to its cytotoxic potency. The cinnamatederivative 48 was found to be substantially less potent (40-50×)suggestive of the requirement for a rigid N² DNA binding subunit.Finally, the agents exhibited a smooth trend of increasing cytotoxicpotency as the size and length of the DNA binding subunits increasedwith iso-CBI-CDPI, and iso-CBI-CDPI₂ displaying the most potentcytotoxic activity in the series exhibiting IC₅₀ values of 200 and 50pM, respectively. (While we were unable to isolate N-BOC-iso-CI (21) andiso-CI-TMI for direct comparison, their seco precursors (±)-19 and(±)-20 exhibited surprisingly potent cytotoxic activity (IC₅₀, L1210=10μM and 6 nM respectively) that likely exceed s that of the ring closedmaterials themselves.)

Findings: iso-CI and iso-CBI were designed to test a key element of thedifferent proposals for the origin of the DNA alkylation sequence withthe duocarmycins and CC-1065. These agents were prepared by applicationof a directed ortho metallation and ortho spirocyclization in animproved synthetic scheme complementary to that reported for CI and CBI.Iso-CBI was found to be 5× less stable than CBI and possess a reactivitycomparable with CC-1065 and duocarmycin A. In addition, nucleophilicaddition occurred at the least substituted cyclopropane carbon with aregioselectivity (40:1) comparable to that of CBI but which exceeds thatof the natural products themselves (6-1.5:1). Comparison of the X-raystructures of iso-CBI and CBI revealed the near identical non-idealconjugation of the cyclopropanes and the exclusive stereoelectronicalignment of the cleaved cyclopropane bond. Consistent with recentobservations, the lower stability of iso-CBI relative to CBI itself canbe attributed to a diminished cross-conjugated vinylogous amidestabilization for which the N²—C^(2a) bond length is diagnostic.^(7.42)Resolution and incorporation of iso-CBI into a full set of duocarmycinand CC-1065 analogs allowed for comparison of their properties and afurther distinguishing test for the origin of the DNA alkylationsequence selectivity. The iso-CBI analogs were highly potent cytotoxicagents exhibiting picomolar IC₅₀'s which correlated with their relativestability. In addition to smoothly following this correlation, theanalogs displayed a smooth trend of increasing cytotoxic potency withthe increasing length in the DNA binding subunit. Analogous to thenatural products, the (S)-enantiomer possessing the absoluteconfiguration of 1-3, proved to be more potent (10-50×) than the(R)-enantiomer. DNA alkylation studies revealed a strong correlationbetween DNA alkylation efficiency and cytotoxic potency as(−)-iso-CBI-TMI was approximately 50-100× less efficient than(+)-CBI-TMI and (+)-duocarmycin SA In addition, both the iso-CI andiso-CBI analogs, with the repositioned C-4 carbonyl, exhibited theidentical sequence selectivity and alkylated the same sites as CBI-TMIand duocarmycin SA derived from adenine N3 addition to the leastsubstituted cyclopropane carbon at comparable reaction rates. This isinconsistent with a proposal that the DNA alkylation selectivity iscontrolled by a sequence-dependent DNA backbone phosphate protonation ofthe C-4 carbonyl for activation of alkylation but is consistent with thenoncovalent binding and steric accessibility model. Finally, this set ofisomeric analogs contains the most significant structural modificationto the alkylation subunit to date, yet remain effective DNA alkylatingagents with properties comparable to the natural products themselves.

Synthesis of Materials

1-(tert-Butyloxycarbonyl))-4-hydroxy-3-[[(methanesulfonyl)oxy]methyl]-2,3-dihydroindole(19): A solution of 18 (22 mg, 0.057 mmol) in 2 mL of 1:1 i-PRO/HF wastreated with 12 N HCl (33 μL, 0.4 mmol) and stirred for 48 h at 25° C.The reaction solution was concentrated under reduced pressure. Flashchromatography (SiO₂, 1.5×10 cm, 40% EtOAc/hexane) afforded recovered 18(8 mg, 41%) and 19 (9.5 mg, 48%) as a white film: ¹H NMR (CDCl₃, 400MHz) δ 7.43 (m, 1H), 7.07 (m, 1H), 6.39 (d, J=8.6 Hz, 1H), 5.44 (br s,1H), 4.55 (dd, J=4.5, 9.8 Hz, 1H), 4.26 (dd, J=8.1, 9.8 Hz, 1H), 4.00(m, 2H), 3.79 (m, 1H); IR (film) ν_(max) 3347, 2977, 1682, 1622, 1602,1467, 1394, 1172, 1145, 948 cm⁻¹; FABHRMS (NBA/CsI) m/z 476.0156(C₁₅H₂₁NO₆S+Cs⁺ requires 476.0144).

4-Hydroxy-3[[(methanesulfonyl)oxy]methyl]-1-[5,6,7-trimethoxyindol-2-yl)carbonyl]-2,3-dihydroindole(20): A sample of 18 (9.0 mg, 0.023 mmol) was dissolved in 3.6 NHCl/EtOAc (2.4 mL) and the solution was stirred for 30 min at 25° C. Thesolvents were removed by a stream of N₂ and the residual salt wasthoroughly dried under high vacuum. The salt was dissolved in anhydrousDMF (1.2 mL) and treated with 37 (7 mg, 0.028 mmol) and EDCI (13 mg,0.07 mmol). The resulting solution was stirred at 25° C. for 3 h underAr. The reaction mixture was then diluted with H₂O (5 mL), and extractedwith EtOAc (3×5 mL). The combined organic layer was dried (Na₂SO₄), andconcentrated under reduced pressure. Flash chromatography (SiO₂, 1.5×10cm, 50% EtOAc/hexane) yielded pure 20 (6.0 mg, 55%) as a white film: ¹HNMR (CDCl₃, 400 MHz) δ 9.33 (br s, 1H), 7.87 (d, J=8.0 Hz, 1H), 7.19 (m,1H), 6.92 (d, J=2.4 Hz, 1H), 6.84 (s, 1H), 6.52 (dd, J=0.5, 8.0 Hz, 1H),4.65 (dd, J=4.3, 10.1 Hz, 1H), 4.58 (dd, J=9.2, 10.8 Hz, 1H), 4.51 (dd,J=3.9, 10.8 Hz, 1H), 4.31 (dd, J=8.4, 10.1 Hz, 1H), 4.05 (s, 3H), 4.01(m, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 2.95 (s, 3H); IR (film) ν_(max)3261, 2938, 1659, 1611, 1462, 1352, 1306, 1282, 1174, 1107 cm⁻¹; FABHRMS(NBA/CsI) m/z 609.0319 (C₂₂H₂₄N₂O₈S+Cs⁺ requires 609.0308).

N-(tert-Butyloxycarbonyl)-4-(methoxymethoxy)-2-naphthylamine (23): Asolution of 22²² (6.67 g, 25.0 mmol) in 125 mL of anhydrous DMF at 0° C.was treated with NaH (1.13 g, 28.0 mmol) in several portions over 5 min.After 10 min, Bu₄NI (0.925 g, 2.5 mmol) was added followed by thedropwise addition of ClCH₂OCH₃ (2.9 mL, 38 mmol). The reaction mixturewas stirred at 25° C. for 5 h before the reaction was quenched by theslow addition of 100 mL of H₂O. The aqueous layer was extracted withEtOAc (3×100 mL). The organic layers were combined, washed with 10%aqueous NaHCO₃ (100 mL), H₂O (4×50 mL), dried (Na₂SO₄), and concentratedunder reduced pressure. Flash chromatography (SiO₂, 4×15 cm, 0-15%EtOAc/hexane gradient) provided 23 (6.0 g, 78%) as a peach coloredsolid: mp 64-66° C., ¹H NMR (CDCl₃, 250 MHz) δ 8.15 (d, J=7.8 Hz, 1H),7.67 (m, 2H) 7.38 (m, 2H), 7.05 (d, J=1.9 Hz, 1H), 6.87 (br s, 1H), 5.34(s, 2H), 3.51 (s, 3H), 1.54 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz). 153.3,152.8, 136.0, 134.8, 127.0, 126.9, 123.7, 122.5, 121.6, 108.0, 101.8,94.6, 80.5, 56.1, 28.2; IR (film) ν_(max) 3334, 2977, 1713, 1634, 1538,1392, 1367, 1248, 1160, 1057 cm⁻¹; FABHRMS (NBA) m/z 303.1463 (C₁₇H₂₁NO₄requires 303.1471). Anal. Calcd for C₁₇H₂₁NO₄: C, 67.31; H, 6.98; N,4.62. Found: C, 67.13; H, 7.18; N, 4.89.

N-(tert-Butyloxycarbonyl)-3-iodo-4-(methoxymethoxy)-2-naphthylamine(24): A solution of 23 (0.435 g 1.43 mmol) in 5.7 mL anhydrous THF wascooled to −25° C. and treated with TMEDA (0.758 mL, 5.0 mmol) followedby n-BuLi (2.29 mL of a 2.5 M solution in hexane, 5.0 mmol) in a slowdropwise manner. The resulting gold solution was stirred for 2 h at −25°C. The reaction mixture was treated with 1-chloro-2-iodoethane (0.37 mL,5.0 mmol) and stirred for 15 min at 25° C. The reaction was diluted withH₂O (40 mL), extracted with Et₂O (3×20 mL), and the combined organicextracts were washed with saturated aqueous NaCl, dried Na₂SO₄) andconcentrated under reduced pressure. Flash chromatography (SiO₂, 2.5×15cm, 0-7% EtOAc/hexane gradient) yielded 24 (490 mg, 80%) as a yellowoil: ¹H NMR (CDCl₃, 400 MHz) δ 8.35 (s, 1H), 8.03 (d, J=12.8 Hz, 1H),7.78 (d, J=12.5 Hz, 1H), 7.42 (m, 2H), 7.14 (br s, 1H), 5.20 (s, 2H),3.74 (s, 3H), 1.56 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ 154.7, 152.6,135.1, 134.8, 127.6, 127.4, 125.1, 125.0, 122.2, 113.0, 100.5, 87.7,81.1, 58.5, 28.4; IR (film) ν_(max) 3391, 2977, 2933, 1732, 1524, 1367,1340, 1276, 1228, 1157 cm⁻¹; FABHRMS (NBA/NaI) m/z 430.0507(C₁₇H₂₀INO₄+H⁺ requires 430.0515).

2-[[N-(tert-Butyloxycarbonyl)-N-(2-propen-1-yl)amino]-3-iodo-4-(methoxymethoxy)naphthalene(25): A solution of 24 (0.490 g, 1.1 mmol) in 36 mL anhydrous DMF wascooled to −10° C., and treated with NaH (69 mg, 1.7 mmol) in smallportions. The resulting suspension was stirred 15 min and treated withneat allyl bromide (0.49 mL, 5.7 mmol) in a slow dropwise manner. Thereaction mixture was warmed to 25° C. and stirred for 1 h. The reactionmixture was quenched with the addition of 5% aqueous NaHCO₃ (50 mL), andthe aqueous layer was extracted with EtOAc (3×20 mL). The combinedorganic extracts were washed with H₂O (5×10 mL) dried (Na₂SO₄), andcondensed under reduced pressure to yield 25 as a 2:1 mixture of amiderotamers as a yellow oil. Flash chromatography (SiO₂, 2.5×15 cm, 10%EtOAc/hexane) yielded 25 (503 mg, 94%) as a colorless oil: ¹H NMR(CDCl₃, 250 MHz) δ 8.16 (m, 1), 7.77 (m, 1H), 7.50 (m, 3H), 5.97 (m, 1),5.23 (m, 2H), 5.07 (m, 2H), 4.56 (m, 1H), 3.80 (m, 1H), 3.72 (s, 3H),1.55 and 1.33 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ 155.6 and 155.4, 154.1and 153.9, 141.3 and 141.1, 133.8 and 133.5, 127.7 and 127.6, 127.1,126.8, 125.2, 124.5, 122.4, 117.8 and 117.3, 100.6, 95.7, 80.6 and 80.3,58.3, 53.6, 52.4, 28.3 and 28.2; IR (film) ν_(max) 2975, 2930, 1703,1581, 1566, 1385, 1366, 1159, 1047 cm⁻¹; FABHRMS (NBA/CsI) m/z 601.9825(C₂₀H₂₄INO₄+Cs⁺ requires 601.9804).

1-(tert-Butyloxycarbonyl)-4-(methoxymethoxy)-3-[[(2′,2′,6′,6′-tetramethylpiperidino)oxy]methyl]-2,3-dihydro-1H-benzo[f]indole(26): A solution of 25 (470 mg, 1.00 mmol) and TEMPO (468 mg, 3.0 mmol)in 43 mL anhydrous benzene was treated with Bu₃SnH (0.283 mL, 1.05mmol). The solution was warmed at 50° C. and an additional 1.05 equiv ofBu₃SnH (0.283 mL, 1.05 mmol) was added twice during the next 30 min.Another 3.0 equiv of TEMPO (468 mg, 3.0 mmol) was added in 10 mLanhydrous benzene, along with an additional 1.05 equiv of Bu₃SnH addedtwice during the next 45 min. After 1.5 h total, the solution was cooledto 25° C., and the volatiles were removed under reduced pressure. Flashchromatography (SiO₂, 2.5×15 cm, 0-12% EtOAc/hexane gradient) provided26 (470 mg, 94%) as a yellow oil: ¹H NMR (CDCl₃, 250 MHz) δ 8.05 (br s,1H), 7.97 (d, J=8.0 Hz, 1H), 7.74 (d, J=7.7 Hz, 1H), 7.36 (m, 2H), 5.24(d, J=5.9 Hz, 1H), 5.16 (d, J=5.9 Hz, 1H), 4.26 (m, 1H), 4.15 (m, 1H),4.01 (m, 1H), 3.81 (m, 2H), 3.63 (s, 3H), 1.61 (s, 9H), 1.23 (s, 3H),1.17 (s, 3H), 1.04 (s, 3H), 1.04 (s, 3H), 0.99 (s, 3H), 1.48-0.89 (m,6H); ¹³C NMR (CDCl₃, 62.5 MHz) δ 152.0, 149.3, 141.1, 135.3, 127.2,125.7, 124.4, 123.4, 122.6, 121.0, 107.0, 99.1, 80.2, 76.0, 59.3, 57.1,51.2, 39.1, 32.6, 27.9, 19.6, 16.6; IR (film) ν_(max) 2974, 2931, 1709,1634, 1446, 1374, 1352, 1332, 1147 cm⁻¹; FABHRMS (NBA/CsI) m/z 631.2168(C₂₉H₄₂N₂O₅+Cs⁺ requires 631.2148).

1-(tert-Butyloxycarbonyl)-3-(hydroxymethyl)-4-(methoxymethoxy)-2,3-dihydro-1H-benzo[f]indole (27): A solution of 26 (220 mg, 0.44 mmol) in 15 mL 3:1:1HOAc/H₂O/THF was treated with Zn powder (1.15 g, 17.6 mmol) and theresulting suspension was warmed at 70° C. under a reflux condenser andwith vigorous stirring for 1 h. The reaction mixture was cooled to 25°C., and the Zn was removed by filtration through Celite with a 25 mLCH₂Cl₂ wash. The volatiles were removed under reduced pressure, and theresulting residue was dissolved in 25 mL of EtOAc and filtered. Thesolution was concentrated under reduced pressure. Flash chromatography(SiO₂, 2.5×10 cm, 304-0% EtOAc/hexane gradient) provided 27 (138 mg,87%) as a colorless oil: ¹H NMR (CDCl₃, 250 MHz) δ 8.05 (br s, 1H), 7.91(d, J=8.0 Hz, 1H), 7.72 (d, J=7.5 Hz, 1H), 7.36 (m, 2H), 5.23 (d, J=6.0Hz, 1H), 5.16 (d, J=6.0 Hz, 1H), 4.10 (m, 1H), 3.94-3.78 (m, 4H), 3.63(s, 3H), 2.04 (d, J=8.4 Hz, 1H), 1.58 (s, 9H); ¹³C NMR (CDCl₃, 125 MHz)δ 152.5, 149.7, 142.0, 136.0, 127.9, 126.4, 124.6, 124.1, 123.1, 121.3,107.8, 100.0, 81.1, 65.1, 57.8, 51.3, 40.7, 28.3; IR (film) ν_(max)3447, 2975, 1704, 1634, 1447, 1353, 1336, 1149 cm⁻¹; FABHRMS (NBA) m/z360.1821 (C₂₀H₂₅NO₅+H⁺ requires 360.1811).

1-(tert-Butyloxycarbonyl)-3-chloromethyl-4-(methoxymethoxy)-2,3-dihydro-1H-benzoylindole(28). From 27: A solution of 27 (55 mg, 0.16 mmol) in 3 mL anhydrousCH₂Cl₂ was treated with CCl₄ (155 μL, 1.6 mmol) and Ph₃P (212 mg, 0.81mmol) and the mixture was stirred at 25° C. for 2 h. The solution wasconcentrated in vaciuo. Flash chromatography (SiO₂, 1.5×15 cm, 0-12%EtOAc/hexane gradient) provided 28 (52 mg, 90%) as a white solid: mp99-101° C; ¹H NMR (CDCl₃, 250 MHz) δ 8.03 (br s, 1H), 7.89 (d, J=8.1 Hz,1H), 7.72 (d, J=7.6 Hz, 1H), 7.37 (m, 2H), 5.24 (d, J=6.1 Hz, 1H), 5.15(d, J=6.1 Hz, 1H), 4.06 (m, 4H), 3.65 (s, 3H), 3.51 (app t, J=10.1 Hz,1H), 1.59 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ 152.3, 150.5, 141.1,136.2, 127.9, 126.6, 124.6, 124.1, 122.4, 121.4, 107.7, 100.0, 81.2,57.5, 52.1, 45.9, 40.8, 28.4; IR (film) ν_(max) 2976, 1704, 1634, 1449,1336, 1147, 1055, 983 cm⁻¹; FABHRMS (NBA) m/z 378.1463 (C₂₀H₂₄Cl NO₄+H⁺requires 378.1472). Anal. Calcd for C₂₀H₂₄INO₄; C, 63.75; H 6.40; N,3.71. Found: C, 63.42; H, 6.11; N, 3.41.

From 29: A solution of 29 (500 mg, 1.0 mmol) in 10 mL anhydrous benzenewas treated with AIBN (15.7 mg, 0.1 mmol) and Bu₃SnH (295 μL, 1.1 mmol)and warmed at 80° C. for 2 h. The reaction mixture was concentratedunder reduced pressure and flash chromatography (SiO₂, 2.5×15 cm, 0-20%EtOAc/hexanes gradient) yielded 28 (360 mg, 96%) as a white solididentical to that described above.

Resolution of (28). A sample of 28 (4.0 mg) in 0.9 mL of 5%i-PrOH/hexane was resolved on a semipreparative Daicel Chiralcel ODcolumn (10 μm, 2×25 cm, 7.0 mL/min flow rate, 3% i-PrOH/hexane). Theeffluent was monitored at 254 nm and the enantiomers eluted withretention times of 29.0 and 35.8 min (α=1.25). The first enantiomer (S)was found to be >99% enantiomerically pure. The second fraction (R) wasreinjected in order to obtain a sample of >99% ee. The fractionscontaining the separated enantiomers were collected and concentrated toafford (+) and (−)-28. (+)-(3S)-28: [α]_(D) ²⁵ +29 (c 0.50, CH₂Cl₂);(−)-(3R)-28: [α]_(D) ²⁵ −30 (c 0.50, CH₂Cl₂).

2-[[N-(tert-Butyloxycarbonyl)-N-(3-chloro-2-propen-1-yl)]amino-3-iodo-4-(methoxymethoxy)naphthalene(29): A solution of 24 (0.480 g, 1.1 mmol) in 11 mL anhydrous DMF wascooled to 0° C., and treated with NaH (67 mg; 2.2 mmol) in smallportions. The resulting suspension was stirred 15 min and treated withneat 1,3-dichloropropene (0.52 mL, 5.5 mmol) in a slow dropwise manner,followed by catalytic Bu₄NI (40 mg, 0.1 mmol). The reaction mixture waswarmed to 25° C. and stirred for 12 h. The reaction mixture was quenchedwith the addition of 5% aqueous NaHCO₃ (50 mL), and the aqueous layerwas extracted with EtOAc (3×20 mL). The combined organic extracts werewashed with H₂O (5×10 mL), dried (Na₂SO₄), and concentrated underreduced pressure to yield 29 as a mixture of rotamers and E and Zalkenes as a yellow oil. Flash chromatography (SiO₂, 2.5 15 cm, 0-20%EtOAc/hexanes gradient) yielded 29 (540 mg, 96%) as a yellow oil: ¹H NMR(CDCl₃, 250 MHz) δ 8.16 (m, 1H), 7.80 (m, 1H), 7.55 (m, 2H), 7.44 (s,1H), 6.11 (m, 2H), 5.25 (d, J=5.6 Hz, 1H), 5.20 (d, J=5.6 Hz, 1H), 4.51(m, 1H), 3.75 (m, 1H), 3.73 (s, 3H), 1.55 and 1.31 (s, 9H); ¹³C NMR(CDCl₃, 62.5 MHz) δ 156.2, 153.9, 140.7, 134.0, 128.7, 127.9, 127.7,127.4, 127.1, 124.7, 122.5, 121.7, 100.7, 80.8, 77.2, 58.5, 49.5, 28.2;IR (film) ν_(max) 2975, 1699, 1565, 1387, 1366, 1328, 1294, 1254, 1162cm⁻¹; FABHRMS (NBA/NaI) m/z 504.0424 (C₂₀H₂₃CIINO₄+H⁺ requires504.0439).

1-(tert-Butyloxycarbonyl-3-chloromethyl)-4-hydroxy-2,3-dihydro-1H-benzo[f]indole(30): A solution of 23 (18 mg, 47.5 μmol) in 1.5 mL of 1:1 i-PrOH/THFwas treated with 12 N HCl (0.20 mL, 0.38 mmol) and the mixture wasstirred at 25° C. for 6 h before the volatiles were removed in vacuo.Flash chromatography (SiO₂, 1.5×15 cm, 0-20% EtOAc/hexane gradient)provided 30 (14.5 mg, 90%) as a pale yellow oil: ¹H NMR (CDCl₃, 250 MHz)δ 7.85 (d, J=8.0 Hz, 1H), 7.71 (d, J=7.8 Hz, 1H), 7.43-7.30 (m, 2H),5.67 (s, 1H), 4.09-3.86 (m, 4H), 3.65 (dd, J=8.0, 10.0 Hz, 1H), 1.59 (s,9H); ¹³C NMR (CDCl₃, 62.5 MHz) δ 147.9, 135.8, 132.7, 128.8, 128.0,126.6, 123.7, 121.2, 119.5, 119.5, 104.4, 77.2, 52.6, 46.4, 34.7, 28.5;IR (film) ν_(max) 3368, 2976, 1668, 1477, 1450, 1369, 1145, 978, 746cm⁻¹; FABHRMS (NBA/CsI) m/z 466.0197 (C₁₈H₂₀ClNO₃+Cs⁺ requires466.0186). (−)-(3S)-30: [α]_(D) ²⁵ −54 (c 0.25, CH₃OH); (+)-(3R)-30:[α]_(D) ²⁵ +51 (c 0.30, CH₃OH).

2-(tert-Butyloxycarbonyl)-1,2,9,9a-tetrahydrocyclopropa[c]benzo[f]indol-8-one(N-BOC-iso-CBI, 31): A solution of 30 (5.0 mg, 0.015 mmol) in 0.6 mLCH₃CN was treated with DBU (2.7 μL, 0.018 mmol) and the mixture wasstirred at 25° C. for 15 min. Flash chromatography (SiO₂, 1.5×10 cm, 10%EtOAc/hexane) afforded 31 (4.3 mg, 96%) as a light golden oil: ¹H NMR(CDCl₃, 400 MHz) δ 7.98 (d, J=7.8 Hz, 1H), 7.50 (dt, J=1.4, 7.8 Hz, 1H),7.28 (d, J=7.8 Hz, 1H), 7.21 (dt, J=1.1, 7.8 Hz, 1H), 6.97 (br s, 1H),3.88 (br d, J=11.2 Hz, 1H), 3.81 (dd, J=4.9, 11.1 Hz, 1H), 2.88 (dt,J=5.5, 7.8 Hz), 1.87 (dd, J=3.2, 7.8 Hz, 1H), 1.53 (s, 9H), 1.41 (dd,J=3.2, 5.5 Hz, 1H); ¹H NMR (C₆D₆, 400 MHz) δ 8.34 (dd, J=0.6, 7.8 Hz,1H), 7.10 (m, 2H), 6.92 (m, 1H), 3.18 (br m, 1H), 2.32 (m, 1H), 1.49(dd, J=3.1, 7.8 Hz, 1H), 1.39 (s, 9H), 0.66 (dd, J=3.1, 5.4 Hz, 1H); ¹³CNMR (CDCl₃, 62.5 MHz) δ 194.7, 154.3, 142.0, 134.3, 127.7, 127.3, 125.9,125.2, 103.2, 77.2, 51.5, 46.1, 32.0, 28.2, 25.7; IR (film) ν_(max)2973, 2927, 1716, 1670, 1635, 1472, 1410, 1324, 1143, 1009 cm⁻¹; UV(THF) λ_(max) (ε) 387 (2700), 302 (6000 sh), 293 (6900), 250 (20400) nm;FABHRMS (NBA/NaI) m/z 298.1450 (C₁₈H₁₉NO₃+H⁺ requires 298.1443).(−)-(8aS,9aS)-31: [α]_(D) ²⁵ −172 (c 0.08, CH₃OH); (+)-(8aR,9aR)-31:[α]_(D) ²⁵ +179 (c 0.12, CH₃OH).

3-Chloro-4-hydroxy-1-(methoxycarbonyl)-2,3-dihydro-1H-benzo[f]indole(32): A solution of 28 (11 mg, 29.1 μmol) was treated with 1.0 mL of 3.6N HCl/EtOAc and the resulting solution was stirred for 30 min at 25° C.The solvent was removed by a stream of N₂ and the residual salt wasdried under vacuum. This salt was suspended in 0.6 mL of anhydrous TBand treated with NaHCO₃ (5.4 mg, 64.1 μmol, 2.2 equiv) and ClCO₂CH₃ (4.5μL, 58.2 μmol, 2.0 equiv) and the mixture was stirred for 3 h at 25° C.Upon completion, the reaction mixture was diluted with 10 mL of H₂O,extracted with EtOAc (3×10 mL), dried (Na₂SO₄) and concentrated invacuo. Flash chromatography (SiO₂, 1.5×15 cm, 0-40% EtOAc/hexanegradient) provided pure 32 (5.5 mg, 65%) as a white solid: ¹H NMR(CDCl₃, 400 MHz) δ 7.86 (d, J=8.2 Hz, 1H), 7.86 (br s, 1H), 7.7, (d,J=8.0 Hz, 1H), 7.42 (m, 1H), 7.35 (m, 1H), 5.80 (s, 1H), 4.11 (m, 2H),4.01-3.87 (m, 5H), 3.63 (app t, J=9.4 Hz, 1H); IR (film) ν_(max) 3379,2956, 1673, 1435, 1372, 1283, 1218 cm⁻¹; FABHRMS (NBA/NaI) m/z 291.0656(M⁺, C₁₅H₁₄ClNO₃ requires 291.0662).

The structure and absolute configuration of (3R)-32 derived from theslower eluting enantiomer of 28 (t_(R)=35.8 min, [α_(D) ²⁵ −9 (c 0.10,CH₃OH)) was obtained from a single-crystal X-ray structure determinationconducted on prisms grown from EtOAc/hexane.

2-(Methoxycarbonyl)-1,2,9,9a-tetrahydrocyclopropa[c]benzo[f]indol-8-one(N—CO₂Me-iso-CBI, 33): A solution of 32 (5.0 mg, 0.017 mmol) in 0.5 mLof CH₃CN was treated with DBU (3.9 μL, 0.025 mmol) and the mixture wasstirred at 25° C. for 15 min. Flash chromatography (SiO₂, 1.5×10 cm,040% EtOAc/hexane gradient) afforded 33 (4.0 mg, 91%) as a pale yellowsolid: ¹H NMR (C₆D₆, 400 MHz) δ 8.33 (d, J=6.0 Hz, 1H), 7.40 (br s, 1H),7.10 (app t, J=6.0 Hz, 1H), 7.03 (d, J=6.0 Hz, 1H), 6.91 (m, 1H), 3.36(s, 3H), 3.17 (m, 1H), 2.93 (m, 1H), 2.30 (dt, J=4.0, 6.0 Hz, 1H), 1.49(dd, J=2.4, 6.4 Hz, 1H), 0.64 (dd, J=2.8, 4.4 Hz, 1H); IR (film) ν_(max)2954, 1731, 1633, 1445, 1323, 1196, 1018 cm⁻¹; UV (THF) λ_(max) (ε) 386(2300), 302 (4500 sh), 293 (5100), 250 (18000) nm; FABHRMS (NBA/NaI) m/z255.0901 (M⁺, C₁₅H₁₃NO₃ requires 255.0895). (+)-(8aR,9aR)-33: [α]_(D) ²⁵+31 (c 0.07, CH₃OH).

The structure of 33 was confirmed with a single-crystal X-ray structuredetermination conducted on plates grown from EtOAc/hexane.³⁷

1,2,9,9a-Tetrahydrocyclopropa[c]benzo[f]indol-8-one (iso-CBI, 34): Asolution of 28 (10.6 mg, 28.1 μmol) was treated with 3.6 M HCl/EtOAc andthe mixture was stirred at 25° C. for 30 min. The volatiles were removedby a stream of N₂ and the residual salt was suspended in 1.0 mL ofdegassed CH₃CN. The suspension was treated with DBU (9.3 μL, 61.8 μmol,2.2 equiv) and stirred under Ar for 20 min at 25° C. in the dark. Flashchromatography (SiO₂, 1.5×15 cm, 40% EtOAc/hexane) provided pure 34 (3.0mg, 55%) as an unstable bright yellow oil: ¹H NMR (C₆D₆, 400 MHz) δ 8.38(d, J=8.0 Hz, 1H), 7.18 (m, 1H), 6.99 (d, J=8.0 Hz, 1H), 6.87 (m, 1H),5.27 (s, 1H), 2.60 (m, 1H), 2.52 (m, 1H), 2.45 (m, 1H), 1.70 (m, 1H),1.00 (m, 1H); IR (film) ν_(max) 3358, 1682, 1594, 1470, 1284, 1262, 1223cm⁻¹; FABHRMS (NBA/NaI) m/z 198.0926 (C₁₃H₁₁NO+H⁺ requires 198.0919).

9,9a-1H-Dihydrocyclopropa[c]benzo[f]indol-3,8-dione (35): A solution of28 (10.6 mg, 28.1 μmol) was treated with 4 M HCl/EtOAc and the mixturewas stirred at 25° C. for 30 min. The volatiles were removed by a streamof N₂ and the residual salt was suspended in 1.0 mL of THF. Thesuspension was treated with 1.0 mL of 5% aqueous NaHCO₃ and stirredexposed to the air for 2 h at 25° C. The bright orange solution wasextracted with EtOAc (3×5 mL), washed with H₂O (2×5 mL) dried (Na₂SO₄),and concentrated in vacuao. Flash chromatography (SiO₂, 1.5×15 cm 60%EtOAc/hexane) provided pure 35 (3.5 mg, 63%) as an orange film: ¹H NMR(C₆D₆, 400 MHz) δ 8.14 (m, 1H), 8.05 (m, 1H), 7.00 (m, 2H), 3.62 (dd,J=6.2, 19.4 Hz, 1H), 3.52 (ddt, J=0.6, 2.2, 19.4 Hz, 1H), 2.55 (ddt,J=2.2, 6.0, 8.3 Hz, 1H), 1.54 (ddd, J=0.6, 3.3, 8.4 Hz, 1H), 0.30 (dd,J=3.3, 5.8 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 191.6, 182.0, 168.9,135.8, 135.6, 135.0, 134.6, 128.4, 127.0, 63.8, 49.7, 35.0, 30.4; IR(film) ν_(max) 2923, 1692, 1680, 1585, 1361, 1262, 1223, 1082 cm⁻¹; UV(CH₃OH), λ_(max) (ε) 271 (5200), 239 (13300) nm; FABHRMS (NBA/NaI) m/z:212.0715 (C₁₃H_(9NO) ₂+H⁺ requires 212.0712).

4-(tert-Butyloxycarbonyl)-1,2,2a,3-tetrahydrofurano[4,3,2-c,d]benzo[f]indole(36): A neat sample of 31 (6.0 mg, 16 μmol) was allowed to sit underroom light at 25° C. for 48 h to yield 36 (6.0 mg, 100%): ¹H NMR (CD₃CN,500 MHz) δ 7.80 (d, J=8.0 Hz, 1H), 7.67 (d, J=7.7 Hz, 1H), 7.35 (m, 1H),7.29 (m, 1H), 7.05 (br s, 1H), 5.22 (dd, J=8.0, 8.5 Hz, 1H), 4.64 (dd,J=9.0, 11.0 Hz, 1H), 4.40 (dd, J=8.5, 10.5 Hz, 1H), 4.22 (m, 1H), 3.88(m, 1H), 1.55 (br s, 9H); IR (film) ν_(max) 2975, 2932, 1699, 1475,1456, 1418, 1353, 1166, 1132 cm⁻¹; FABHRMS (NBA/NaI) m/z 297.1451 (M⁺,C₁₈H₁₉NO₃ requires 297.1365).

3-Chloromethyl-4-hydroxy-1-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[f]indole(43): A sample of 28 (8.4 mg, 0.022 mmol) was treated with 1.0 mL of 3.6N HCl/EtOAc and the resulting solution was stirred for 30 min at 25° C.The solvent was removed by a stream of N₂ and the residual salt wasdried under vacuum. This salt was dissolved in 0.9 mL of anhydrous DMFand treated with 5,6,7-trimethoxyindole-2-carboxylic acid¹⁰ (6.8 mg,0.026 mmol) and EDCI (12.7 mg, 0.067 mmol) and the mixture was stirredfor 3 h at 25° C. Upon completion, the reaction mixture was diluted with20 mL of EtOAc, washed with H₂O (5×3 mL), dried (Na₂SO₄), andconcentrated in vacuo. Flash chromatography (SiO₂, 1.5×15 cm, 0-40%EtOAc/hexane gradient) provided 43 (11.2 mg, 91%) as a white solid: ¹HNMR (CDCl₃, 400 MHz) δ 9.41 (s, 1H), 8.35 (s, 1H), 7.88 (d, J=8.4 Hz,1H), 7.82 (d, J=7.8 Hz, 1H), 7.45 (m, 1H), 7.41 (m, 1H), 6.96 (d, J=2.3Hz, 1H), 6.85 (s, 1H), 5.95 (br s, 1H), 4.58 (m, 2H), 4.07 (m, 2H), 4.07(s, 3H), 3.94 (s, 3H), 3.90 (s, 3H), 3.64 (dd, J=10.3, 12.0 Hz, 1H); ¹³CNMR (acetone-d₆, 400 MHz) δ 193.6, 161.1, 151.0, 149.7, 143.4, 141.5,136.7, 131.6, 128.8, 127.3, 126.5, 124.7, 124.5, 124.0, 121.9, 115.3,107.6, 107.2, 99.0, 61.44, 61.37, 56.4, 54.9, 46.6, 41.7; IR (film)ν_(max) 3405, 3301, 2937, 1594, 1446, 1312, 1228, 1106 cm⁻; FABHRMS(NBA/CsI) m/z 467.1358 (C₂₃H₂₃ClN₂O₅+H⁺ requires 467.1374). (−)-(3S)-43:[α]_(D) ²⁵ −4 (0.22, CHCl₃); (+)-(3R)-43: [α]_(D) ⁵+4 (c 0.35, CHCl₃).

2-[(5,6,7-Trimethoxyindol-2-yl)carbonyl]-1,2,9,9a-tetrahydrocyclopropa[c]benzo[f]indol-8-one(iso-CBI-TMI, 44): A solution of 43 (5.7 mg, 0.012 mmol) in 0.5 mL ofCH₃CN was treated with DBU (2.2 μL, 0.05 mmol) and stirred at 25° C. for15 min. Flash chromatography (SiO₂, 1.5×10 cm, 50% EtOAc/hexane)provided pure 44 (4.2 mg, 94%) as a light golden oil: ¹H NMR (C₆D₆, 400MHz) δ 9.65 (s, 1H), 8.36 (m, 1H), 7.76 (s, 1H), 7.10 (m, 1H), 7.03 (m,1H), 6.94 (m, 1H), 6.77 (s, 1H), 6.45 (d, J=2.2 Hz, 1H), 3.78 (s, 3H),3.70 (s, 3H), 3.54 (s, 3H), 3.52 (d, J=9.9 Hz, 1H), 3.26 (dd, J=5.2, 9.9Hz, 1H), 2.43 (m, 1H), 1.57 (dd, J=3.3, 7.8 Hz, 1H), 0.70 (dd, J=3.3,5.4 Hz, 1H); ¹³C NMR (adetone-d₆, 400 MHz) δ 194.5, 191.8, 161.7, 151.4,147.1, 143.1, 142.0, 140.4, 135.6, 131.3, 129.4, 128.9, 127.3, 126.9,126.7, 125.0, 112.7, 108.1, 107.6, 99.3, 63.7, 63.6, 57.0, 54.3, 40.6;IR (film) ν_(max) 3295, 2934, 1667, 1651, 1409, 1306, 1279, 1109 cm⁻¹;UV (CH₃OH) λ_(max) 390 (ε 7100), 332 (E 14100), 245 nm (ε 19100);FABHRMS (NBA/NaI) m/z 431.1615 (C₂₅H₂₂N₂O₅+H⁺ requires 431.1607).(−)-(8aS,9aS)-44: [α]_(D) ²⁵ −41 (c 0.13, CH₃OH); (+)-(8aR,9aR)-44:[α]_(D) ²⁵ +38 (c 0.08, CH₃OH).

3-Chloromethyl)-4-hydroxy-1-[(5-methoxyindol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[f]indole(45): Flash chromatography (SiO₂, 1.5×10 cm, 0-40% EtOAc/hexanegradient) afforded pure 45 (95%) as a clear oil: ¹H NMR (CDCl₃, 400 MHz)δ 9.38 (br s, 1H), 8.35 (s, 1H), 7.93 (d, J=7.6 Hz, 1H), 7.82 (d, J=7.9Hz, 1), 7.41 (m, 2H), 7.36 (d, J=9.0 Hz, 1H), 7.11 (d, J=2.2 Hz, 1H),7.01 (m, 2H), 6.43 (s, 1H), 4.63 (d, J=5.4 Hz, 2H), 4.11 (m, 2H), 3.86(s, 3H), 3.64 (m, 1H); IR (film) ν_(max) 3287, 2927, 1665, 1596, 1518,1445, 1402, 1389, 1290, 1231, 1167 cm⁻¹; FABHRMS (NBA/NaI) m/z 406.1071(C₂₅H₁₉ClN₂O₃ requires 406.1084). (−)-(3S)-45: [α]_(D) ²⁵ −26 (c 0.07,CH₃OH); (+)-(3R)-45: [α]_(D) ²⁵ +25 (c 0.04, CH₃OH).

2-[(5-Methoxyindol-2-yl)carbonyl]-1,2,9,9a-tetrahydrocyclopropa[c]benzo[f]indol-8-one(46): Flash chromatography (SiO₂, 1.5×5 cm, 40% EtOAc/hexane) afforded46 (85%) as a light golden oil: ¹H NMR (C₆D₆, 400 MHz) δ 9.15 (s, 1H),8.38 (m, 1H), 7.80 (s, 1H), 7.12-7.05 (m, 4H), 6.94 (ddd, J=1.6, 6.8,8.3 Hz, 1H), 6.85 (app dt, J=0.8, 8.6 Hz, 1H), 6.39 (d, J=1.6 Hz, 1H),3.51 (s, 3H), 3.48 (d, J=10.0 Hz, 1H), 3.21 (dd, J=5.2, 10.0 Hz, 1H),2.42 (dt, J=5.3, 7.8 Hz, 1H), 1.56 (dd, J=3.3, 7.8 Hz, 1H), 0.68 (dd,J=3.3, 5.3 Hz, 1H); IR (film) ν_(max) 3303, 2927, 1667, 1643, 1597,1518, 1408, 1277, 1030, 1013 cm⁻¹; FABHRMS (NBA/NaI) m/z 371.1388(C₂₃H₁₈N₂O₃+H⁺ requires 371.1396). (−)-(8aS,9aS)-46: [α]_(D) ²⁵ −66 (c0.04, CH₃OH); (+)-(8aR,9aR)-46: [α]_(D) ²⁵ +71 (c 0.03, CH₃OH).

3-Chloromethyl)-4-hydroxy-1-[((E)-3-(2-methoxyphenyl)propenyl)carbonyl]-2,3-dihydro-1H-benzo[f]indole(47): Flash chromatography (SiO₂, 1.5×10 cm, 0-40% EtOAc/hexanegradient) afforded 47 (87%) as a pale yellow solid: ¹H NMR (CDCl₃, 400MHz) δ 8.40 (br s, 1H), 7.87 (m, 3H), 7.40 (m, 2H), 7.31 (m, 1H), 7.22(m, 1H), 7.20 (s, 1H), 6.93 (m, 2H, 5.81 (m, 1H), 4.36 (m, 2H), 4.09 (n,2H), 3.85 (s, 3H), 3.65 (m, 1H); IR (film) ν_(max) 3266, 2963, 1659,1598, 1445, 1377, 1269, 1158, 1048 cm⁻¹; FABHRMS (NBA/NaI) m/z 394.1217(C₂₅H₂₀ClNO₃+H⁺ requires 394.1210). (−)-(3S)-47: [α]_(D) ²⁵ −18 (c 0.15,CH₃OH); (+)-(3R)-47: [α]_(D) ²⁵ +22 (c 0.04, CH₃OH).

2-[((E)-3-(2-Methoxyphenyl)propenyl)carbonyl]-1,2,9,9a-tetrahydrocyclopropa[c]benzo[f]indol-1-one(48): Flash chromatography (SiO₂, 1.5×5 cm, 40% EtOAc/hexane) afforded48 (83%) as a light golden oil: ¹H NMR (C₆D₆, 400 MHz) rotamers δ 8.39(d, J=7.5 Hz, 1H), 8.09 (d, J=15.4 Hz, 1H), 7.15-6.95 (m, 8H), 6.76(ddd, J=1.0, 2.5, 8.1 Hz, 1H), 3.703.50 (m, 1H), 3.31 (s, 3H), 3.00-2.80(m, 1H), 2.35 (m, 1H), 1.63 (m, 1H), 0.73 (m, 1H); IR (film) ν_(max)2926, 1673, 1626, 1471, 1408, 1383, 1265, 1156, 1092, 1044, 1009 cm⁻¹;FABHRMS (NBA/NaI) m/z 357.1;60 (M⁺, C₂₅H₁₉INO₃ requires 357.1365).(−)-(8aS,9aS)-48: [α_(D) ²⁵ −21 (c 0.08, CH₃OH); (+)-(8aR,9aR)-48:[α]_(D) ²⁵ +23 (c 0.03, CH₃OH).

3-Chloromethyl-4-hydroxy-1-{[5-[N-(indol-2-yl)carbonyl]aminoindol-2-yl]carbonyl}-2,3-dihydro-1H-benzo[f]indole(49): Flash chromatography (SiO₂, 1.5×10 cm, 40-80% EtOAc/hexanegradient) afforded 49 (74%) as a light brown solid: ¹H NMR (DMF-d₇, 400MHz) δ 11.76 (s, 2H), 10.30 (s, 1H), 10.28 (s, 1H), 8.41 (s, 1H), 8.29(s, 1H), 8.25 (d, J=8.0 Hz, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.71 (dd,J=2.8, 9.2 Hz, 1H), 7.69 (d, J=9.6 Hz, 1H), 7.59 (d, J=8.6 Hz, 2H), 7.52(s, 1H), 7.46 (m, 1H), 7.40 (m, 1H), 7.30 (d, J=1.6 Hz, 1H), 7.25 (m,1H), 7.09 (m, 1H), 4.83 (m, H), 4.70 (dd, J=2.4, 10.4 Hz, 1H), 4.30 (n,1H), 4.16 (dd, J=2.8, 11.2 Hz, 1H), 3.98 (dd, J=8.8, 11.2 Hz, 1H); IR(film) ν_(max) 3280, 2929, 1660, 1650, 1594, 1519, 1443, 1389, 1314,1250, 1098, 749 cm⁻¹; FABHRMS (NBA/CsI) m/z 667.0529 (C₃₁H_(21ClN)₄O₃+Cs⁺ requires 667.0513). (−)-(3S)-49: [α]_(D) ²⁵ −18 (c 0.10, CH₃OH);(+)-(3R)-49: [α]_(D) ²⁵ +21 (c 0.06, CH₃OH).

2-{[5-N-(indol-2-yl)carbonyl]aminoindol-2-yl]carbonyl}-1,2,9,9a-tetrahydrocyclopropa[c]benzo[f]indol-8-one(iso-CBI-indole₂, 50): Flash chromatography (SiO₂, 1.5×10 cm, 10%DMF/toluene) afforded 50 (88%) as a light golden solid: ¹H NMR (DMF-d₇,400 MHz) δ 11.75 (s, 1H), 11.72 (s, 1H), 10.28 (s, 1H), 8.37 (s, 1H),7.96 (d, J=7.9 Hz, 1H), 7.73-7.64 (m, 3H), 7.58 (t, J=8.1 Hz, 2H), 7.50(m, 2H), 7.35 (m, 2H), 7.23 (m, 2H), 7.08 (t, J=7.3 Hz, 1H), 4.57 (m,2H), 3.09 (dt, J=5.5, 7.7 Hz, 1H), 1.86 (dd, J=3.1, 7.7 Hz, 1H), 1.77(dd, J=3.2, 5.5 Hz, 1H); IR (film) ν_(max) 3289, 2927, 1668, 1652, 1557,1520, 1409, 1313 cm⁻¹; FABHRMS (NBA/CsI) m/z 499.1752 (C₃₁H₂₂N₄O₃+H⁺requires 499.1770). (−)-(8aS,9aS)-50: [α]_(D) ²⁵ +21 (c 0.10, DMF);(+)-(8aR,9aR)-50: [α]_(D) ²⁵ +21 (c 0.06, DMF).

1-[(3-Carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indol-7-yl)carbonyl]-3-chloromethyl-4-hydroxy-2,3-1H-benzo[f]indole(51): Flash chromatography (SiO₂, 1.5×10 cm, 10-50% DMF/toluenegradient) afforded pure 51 (80%) as a yellow solid: ¹H NMR (DMF-d₇, 400MHz) δ 11.64 (s, 1H), 10.32 (s, 1H), 8.28 (s, 1H), 8.25 (d, J=8.4 Hz,1H), 8.16 (d, J=8.9 Hz, 1H), 7.81 (d, J=8.1 Hz, 1H), 7.46 (m, 1H), 7.39(m, 2H), 7.15 (d, J=1.7 Hz, 1H), 6.13 (s, 2H), 4.82 (m, 1H), 4.68 (dd,J=2.7, 10.8 Hz, 1H), 4.30 (m, 1H), 4.15 (m, 3H), 3.97 (dd, J=8.3, 10.8Hz, 1H), 3.39 (m, 2H); IR (film) ν_(max) 3337, 2924, 1662, 1652, 1513,1456, 1441, 1400, 1344, 1272 cm⁻¹; ESIMS m/z 461 (M+H⁺, H₂₅H₂₁ClN₄O₃requires 461). (−)-(3S)-51: [α]_(D) ²⁵ −21 (c 0.18, DMF); (+)-(3R)-51:[α]_(D) ²⁵ +22 (c 0.13, DMF).

2-[(3-Carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indol-7-yl)carbonyl]-2,9,9a-tetrahydrocyclopropa[c]benzo[f]indol-8-one(iso-CBI-CDPI₁, 52): Flash chromatography (SiO₂, 1.5×10 cm, 30-50%DMF/toluene gradient) afforded 52 (81%) as a brown solid: ¹H NMR(DMF-d₇, 400 MHz) δ 11.60 (s, 1H), 8.15 (d, J=8.8 Hz, 1H), 7.95 (d,J=7.6 Hz, 1H), 7.66 (t, J=7.4 Hz, 1H), 7.49 (d, J=7.8 Hz, 1H), 7.35 (m,3H), 7.10 (s, 1H), 6.13 (s, 2H), 4.55 (m, 2H), 4.13 (t, J=8.8 Hz, 2H),3.36 (t, J=9.1 Hz, 2H), 3.09 (m, 1H), 1.85 (dd, J=2.9, 7.6 Hz, 1H), 1.76(dd, J=3.3, 5.4 Hz, 1H); IR (film) ν_(max) 3346, 2927, 1667, 1651, 1505,1435, 1408, 1343, 1279, 1098 cm⁻¹; ESIMS m/z 425 (M+H⁺, C₂₅H₂₀N₄O₃requires 425). (−)-(8aS,9aS)-52: [α]_(D) ²⁵ −13 (c 0.06, DMF);(+)-(8aR,9aR)-52: [α]_(D) ²⁵ +12 (c 0.07, DMF).

1-{[3-[N-(3-Carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indol-7-yl)carbonyl]-1,2-dihydro-3H-pyrrolo[3,2-e]indol-7-yl]carbonyl}-3-chloromethyl-4-hydroxy-2,3-dihydro-1H-benzo[f]indole(53): Flash chromatography (SiO₂, 1.5×15 cm, 10-50% DMF/toluenegradient) afforded pure 53 (32%) as a red solid: ¹H NMR (DMF-d₇, 400MHz) δ 11.85 (s, 1H), 11.54 (s, 1H), 10.33 (s, 1H), 8.39 (m, 1H), 8.30(s, 1H), 8.26 (d, J=8.4 Hz, 1H), 8.13 (d, J=8.9 Hz, 1H), 7.82 (d, J=8.0Hz, 1H), 7.48 (m, 2H), 7.38 (m, 2H), 7.31 (s, 1H), 7.08 (s, 1H), 6.10(s, 2H), 4.86 (m, 1H), 4.73 (m, 3H), 4.31 (m, 1H), 4.15 (m, 3H), 3.99(dd, J=8.2, 10.7 Hz, 1H), 3.55 (m, 2H), 3.39 (m, 2H); IR (film) ν_(max)3338, 2956, 2927, 1727, 1659, 1650, 1604, 1510, 1402, 1365, 1286 cm⁻¹;ESIMS m/z 645/647 (M+H⁺, C₃₆H₂₉ClN₆O₄ requires 645/647). (−)-(3S)-53:[α]_(D) ²⁵ −19 (c 0.10, DMF); (+)-(3R)-53; [α]_(D) ²⁵ +21 (c 0.08, DMF).

2-{[3-[N-(3-Carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indol-7-yl)carbonyl]-1,2-dihydro-3H-pyrrolo[3,2-e]indol-7-yl]carbonyl}-1,2,9,9a-tetrahydrocyclopropa[c]benzo[f]indol-1-one(iso-CBI-CDPI₂, 54): Flash chromatography (SiO₂, 1.5×10 cm, 50%DMF/toluene) afforded 53 (59%) as a brown solid: ¹H NMR (DMF-d₇, 400MHz) δ 1.80 (s, 1H), 11.53 (s, 1H), 8.37 (m, 1H), 8.13 (d, J=8.9 Hz,2H), 7.65 (m, 1H), 7.49 (d, J=7.8 Hz, 1H), 7.47 (d, J=9.1 Hz, 1H), 7.36(m, 3H), 7.24 (d, J=1.5 Hz, 1H), 7.07 (s, 1H), 6.12 (s, 2H), 4.74 (t,J=8.4 Hz, 2H), 4.58 (m, 2H), 4.14 (t, J=8.9 Hz, 2H), 3.52 (m, 2H), 3.38(t, J=8.8 Hz, 2H), 3.11 (m, 1H), 1.87 (dd, J=3.1, 7.8 Hz, 1H), 1.78 (dd,J=3.1, 5.5 Hz, 1H); IR (film) ν_(max) 3293, 2926, 1665, 1612, 1581,1503, 1431, 1409, 1363, 1344, 1278, 1185 cm⁻¹; ESIMS m/z 607 (M—T⁺,C₃₆H₂₈N₆O₄ requires 607). (−)(8aS,9aS)-(54: [α]_(D) ²⁵ −34 (c 0.05,DMF); (+)-(8aR,9aR)-54: [α]_(D) ²⁵ ×33 (c 0.03, (DMF).

Solvolysis Regioselectivity:1-(tert-Butyloxycarbonyl)-4-hydroxy-3-(methoxymethyl)-2,3-dihydro-1H-benzo[f]indole(62): A solution of 31 (7.7 mg, 0.026 mmol) in 2.5 mL of CH₃OH wascooled to 0° C., and CF₃SO₃H in CH₃OH (311 μL, 0.01 N, 0.12 equiv) wasadded. After slowly warming to 25° C. over 17 h, the reaction wasquenched by the addition of NaHCO₃ (2.1 mg), filtered through Celite andconcentrated under reduced pressure. Flash chromatography (SiO₂, 1.5×15cm, 0-40% EtOAc/hexane gradient) yielded the major isomer 62 (8.0 mg,94%) as a white film: ¹H NMR (CDCl₃, 400 MHz) δ 9.05 (s, 1H), 8.12 (d,J=6.4 Hz, 1H), 7.80 (m, 1H), 7.65 (s, 1H), 7.37 (m, 1H), 7.29 (m, 1H),4.17 (m, 1H), 3.83 (m, 1H), 3.77 (dd, J=2.8, 6.4 Hz, 1H), 3.60 (dd,J=6.8, 8.8 Hz, 1H), 3.54 (s, 3H), 3.49 (m, 1H), 1.56 (s, 9H); IR (film)ν_(max) 3233, 2975, 1704, 1644, 1435, 1348, 1148, 1026, 956 cm⁻¹;FABHRMS (NBA/NaI) m/z 352.1535 (C₁₉H₂₃NO₄+Na⁺ requires 352.1525).

For the minor isomer 63 (<2% by ¹H NMR of the crude reaction mixture):¹H NMR (CDCl₂, 500 MHz) δ 8.10 (m, 1H), 7.95 (d, J=6.5 Hz, 1H), 7.73 (m,1H), 7.38 (m, 1H), 7.33 (m, 1H), 7.24 (m, 1H), 4.13 (m, 1H), 3.98 (s,3H), 3.92 (m, 1H), 3.81 (m, 2H), 3.50 (m, 1H), 1.57 (s, 9H); FABHRMS(NBA/NaI) m/z 352.1534 (C₁₉H₂₃NO₄+Na⁺ requires 352.1525).

Preparation of Authentic 62: A solution of 27 (17.3 mg, 0.05 mmol) in1.6 mL anhydrous DMF was treated with NaH (4.3 mg, 0.14 mmol) in smallportions and the resulting suspension was stirred for 15 min at 0° C.Methyl iodide (18 μL, 0.29 mmol) was added neat and the resultingmixture warmed to 25° C. over 2 h. The reaction mixture was quenchedwith the addition of 5% aqueous NAHCO₃ (10 mL), and the aqueous layerwas extracted EtOAc (3×5 mL). The combined organic extracts were washedwith H₂O (5×5 mL), dried (Na₂SO₄) and concentrated under reducedpressure. The crude mixture was subjected to flash chromatography (SiO₂,1.5×15 cm, 10% EtOAc-hexanes) to yield the intermediate methyl ether 64(14.0 mg, 78%) as a colorless oil. 64 was subjected to the selective MOMdeprotection reaction and purification as detailed for 30 to yield themajor solvolysis product 62, identical in all aspects.

Solvolysis Reactivity: N-BOC-iso-CBI (31, 0.2 mg) was dissolved in CH₃OH(1.5 mL) and mixed with pH 3 aqueous buffer (1.5 mL). The buffercontained 4:1:20 (v/v/v) 0.1 M citric acid, 0.2 M Na₂HPO₄, and H₂O,respectively. The solvolysis solution was sealed and kept at 25° C.protected from light. The UV spectrum was measured at regular intervalsevery 1 h during the first 24 h, every 4 during the next 72 h, and every12 h for an additional week. The decrease in the long wavelengthabsorption at 397 nm was monitored, FIG. 3. The solvolysis rate constant(k=6.98×10⁻⁶ s⁻¹) and the half-life (t_(1/2)=28 h) were calculated fromdata recorded at the long wavelength from the least-squares treatment(r=0.99) of the slope of the plot of time versusln[(A_(f)-A_(i))/(A_(f)-A)] (FIG. 9).

Similarly, N—CO₂Me-iso-CBI (33, 0.2 mg) was dissolved in CH₃OH (1.5 mL)and mixed with pH 3 aqueous buffer (1.5 mL). The solvolysis solution wassealed and kept at 25° C. protected from light. The UV spectrum wasmeasured at regular intervals every 1 h during the first 24 h, every 4 hduring the next 72 h, and every 12 h for an additional week. Thedecrease in the long wavelength absorption at 395 nm was monitored, FIG.3. The solvolysis rate constant (k=6.40×10⁻⁶ s⁻¹) and the half-life(t_(1/2)=30 h) were determined as detailed above (r=0.99).

Similarly, 35 (0.2 mg) was dissolved in CH₃OH (1.5 mL) and mixed with pH3 aqueous buffer (1.5 mL). The solvolysis solution was sealed and keptat 25° C. protected from light. The UV spectrum was measured at regularintervals every 1 h during the first 12 h, every 2 h for the next 24 h,and every 4 h for an additional day. The decrease in the shortwavelength absorption at 239 nm was monitored, FIG. 9. The solvolysisrate constant (k=3.80×10⁻⁵ s⁻¹) and the half-life (t_(1/2)=5 h) weredetermined as detailed above (r=0.99).

Isolation, Characterization and Quantitation of the Thermally Released(−)-iso-CBI-TMI Adenine Adduct 66: (−)-iso-CBI-TMI (44, 1.0 mg, 2.32μmol) in 500 μL of DMSO was added to a solution of calf thymus DNA(Sigma, 220 mg, ca. 150 bp) in 10 mM sodium phosphate buffer (13 mL, pH7.0) in 50 mL centrifuge tube. The mixture was cooled at 4° C. for 72 h,and then extracted with EtOAc (3×10 mL) to remove hydrolyzed, rearranged(65) or unreacted 44. UV and HPLC assay of the EtOAc extracts revealedno unreacted 44 (0%) and <5% rearranged 65. The centrifuge tubecontaining the aqueous DNA layer was sealed with Teflon tape and warmedat 100° C. for 30 min. The resulting solution was cooled to 25° C. andextracted with EtOAc (3×10 mL). The combined organic layer was dried(Na₂SO₄) and concentrated under reduced pressure to afford a yellowsolid. Chromatography (SiO₂, 0.5×3 cm, 0-7% CH₃OH/CHCl₃ gradientelution) afforded (+)-66 as a pale yellow solid (1.25 mg, 1.31 mgtheoretical, 95%, 90-95% in four runs) contaminated with a smallimpurity (<5% by HPLC). For 66: [α]_(D) ²⁵ +28 (c 0.06, CH₃OH);R_(f)=0.3 (SiO₂, 10% CH₃OH/CHCl₃); ¹H NMR (acetone-d₆, 400 MHz) δ 10.37(s, 1H, N1′-H), 8.58 (s, 1H, ArOH), 8.31 (d, J=7.9 Hz, 1H, Ar—H), 8.22(s, 1H, Ade-C8-H), 8.07 (s, 1H, Ade-C2-H), 7.78 (d, J=7.9 Hz, 1H, Ar—H),7.49 (br s, 1H, Ar—H), 7.45 (ddd, J=1.3, 6.8, 7.9 Hz, 1H, Ar—H), 7.38(ddd, J=1.3, 6.8, 7.9 Hz, 1H, Ar—H), 7.10 (d, J=2.3 Hz, C3′-H), 6.93 (s,1H, C5′-H), 4.92 (dd, J=10.6, 1.9 Hz, 1H, CHH-Ade), 4.89 (dd, J=14.5,7.0 Hz, 1H, CHHNCO), 4.74 (dd, J=14.5, 7.6 Hz, 1H, CHHNCO), 4.73 (dd,J=10.8, 2.2 Hz, 1H, CHH-Ade), 4.37 (m, 1H, CH₂CHCH₂), 4.04 (s, 3H,OCH₃), 3.87 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃); ¹H NMR (DMSO-d₆+1% d-TFA,400 MHz) δ 11.40 (s, 1H, N1′-H), 9.28 (br s, 1H, NHH), 8.78 (br s, 1H,NHH), 8.45 (s, 1H, Ade-C8-H), 8.40 (s, 1H, Ade-C2-H), 8.07 (d, J=7.2 Hz,1H, Ar—H), 8.05 (br s, 1H, ArOH), 7.76 (d, J=7.2 Hz, 1H, Ar—H), 7.43(dd, J=6.4, 6.4 Hz, 1H, Ar—H), 7.35 (dd, J=6.4, 6.4 Hz, 1H, Ar—H), 6.95(s, 1H, C3′-H), 6.90 (s, 1H, C5′-H), 4.72 (dd, J=10.8, 5.6 Hz, 1H,CHH-Ade), 4.68 (dd, J=10.8, 5.6 Hz, 1H, CHH-Ade), 4.55 (dd, J=9.0, 9.0Hz, 1H, CHHNCO), 4.54 (br d, J=9.0 Hz, 1H, CHHNCO), 4.36 (m, 1H), 3.93(s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃); ¹³C NMR (150 MHz,acetone-d₆) δ 170.5, 161.0, 156.9, 152.2, 151.2, 151.0, 150.0, 143.9,142.9, 141.4, 140.0, 136.7, 131.8, 128.4, 127.2, 126.4, 124.7, 124.6,124.1, 123.0, 114.2, 107.3, 106.1, 98.9, 61.5, 61.4, 57.5, 56.5, 56.4,39.8; IR (film) ν_(max) 3251, 2911, 2850, 1684, 1647, 1458, 1312, 1200,1024 cm⁻¹; UV (CH₃OH) λ_(max) 331 (ε 12400), 300 (ε 12600), 240 nm(ε17700); FABHRMS (NBA) m/z 566.2074 (M+H⁺, C₃₀H₂₇N₇O₅ requires566.2083).

HPLC t_(R) (4×250 nm column, 1.0 mL/min, 35% CH₃CN/0.05 N aqueousHCO₂NH₄) were 44 (21.6 min), 65 (39.0 min), solvolysis product (24.0min), and 66 (17.5 min).

Methoxymethyl 3-Nitrophenyl Ether (11): A solution of 3-nitrophenol(5.00 g, 36 mmol) in 100 mL of anhydrous DMF at 0° C. was treated withNaH (2.16 g 54 mmol) in several portions over 5 min. After 10 min, Bu₄NI(1.33 g, 3.6 mmol) was added and followed by dropwise addition ofClCH₂OCH₃ (4.1 mL, 54 mmol). The reaction mixture was stirred at 25° C.for 21 h before being quenched with the slow addition of 100 mL of H₂O.The aqueous layer was extracted with EtOAc (3×100 mL). The organiclayers were combined, washed with 10% aqueous NaHCO₃ (100 mL), H₂O (4×50mL), dried (Na₂SO₄), and concentrated under reduced pressure. Flashchromatography (SiO₂, 4×15 cm, 10% EtOAc/hexane) provided 11 (5.83 g,89%) as a pale yellow solid: ¹H NMR (CDCl₃, 250 MHz) δ 7.86 (m, 2H),7.34 (m, 2H), 5.22 (s, 2H), 3.46 (s, 3H); ¹³C NMR (CDCl₃, 62.5 MHz) δ157.5, 148.9, 129.9, 122.6, 116.5, 110.9, 94.4, 56.1; IR (film) ν_(max)3099, 2959, 2829, 1619, 1584, 1537, 1349, 1237, 1153, 1081 cm⁻¹; FABHRMS(NBA/NaI) m/z: 206.0430 (C₈H₉NO₄+Na⁺ requires 206.0429). Anal. Calcd forC₈H₉NO₄: C, 52.46; H, 4.95; N, 7.65. Found: C, 52.37; H, 4.89; N, 7.61.

3-Aminophenyl Methoxymethyl Ether (12): A solution of 11 (5.68 g, 31mmol) in 310 mL moist ether (8:2:1 Et₂O/EtOH/H₂O) was cooled to 0° C.,and treated with freshly prepared Al—Hg (5.2 g Al, 217 mmol) in small1×1 cm pieces. The reaction mixture was stirred vigorously for 0.5 h at0° C., and 1 h at 25° C. The reaction mixture was filtered throughCelite, and the Celite was washed thoroughly with Et₂O (5×50 mL). Thefiltrate was washed with saturated aqueous NaCl (300 mL), dried(Na₂SO4), and concentrated under reduced pressure to afford 12 (4.23 g,89%) as a golden oil, which was immediately carried on to the next stepwithout further purification. For 12: ¹H NMR (CDCl₃, 250 MHz) δ 7.05 (t,J=8.0 Hz, 1H), 6.38 (m, 3H), 5.12 (s, 2H), 3.70 (br s, 2H), 3.46 (s,3H); ¹³C NMR (CDCl₃, 250 MHz) δ 158.2, 147.7, 129.9, 108.7, 106.0,102.9, 94.0, 55.7; IR (film) ν_(max) 3452, 3367, 2955, 2900, 1623, 1601,1494, 1287, 1147, 1074, 1009 cm⁻; FABHRMS (NBA/NaI) m/z 153.0786(C₈H₁₁NO₂ requires 153.0790).

[N-(tert-Butyloxycarbonyl)amino]-3-(methoxymethoxy)benzene (13): Asolution of crude 12 (4.13 g, 27 mmol) in 135 mL anhydrous THF wastreated with BOC₂O (12.14 g, 54 mmol) and the reaction mixture waswarmed at reflux (65° C.) for 18 h. The solvents were removed underreduced pressure, and flash chromatography (SiO₂, 4×15 cm, 20%EtOAc/hexane) provided pure 13 (6.83 g, 100%) as a yellow oil: ¹H NM(CDCl₃, 250 MHz) δ 7.15 (m, 2H), 6.94 (m, 1H), 6.68 (m, 1H), 6.58 (br s,1H), 5.13 (s, 2H), 3.44 (s, 3H), 1.50 (s, 9H); ¹³C NMR (CDCl₃, 62.5 MHz)δ 157.7, 152.5, 139.5, 129.6, 111.9, 110.7, 106.6, 94.2, 80.5, 55.9,28.2; IR (film) ν_(max) 3337, 2977, 1728, 1605, 1537, 1236, 1153, 1015cm⁻¹; FABHRMS (NBA/NaI) m/z 276.1203 (C₁₃H₁₉NO₄+Na⁺ requires 276.1212)

[N-(tert-Butyloxycarbonyl)amino]-2-iodo-3-(methoxymethoxy)benzene (14):A solution of 13 (124 mg, 0.49 mmol) in 2.0 mL anhydrous THF was cooledto −20° C. and treated with TMEDA (0.26 mL, 1.71 mmol) followed byn-BuLi (0.69 mL of a 2.5 M solution in hexanes, 1.71 mmol) in a slowdropwise manner. The resulting gold solution stirred for 4 h at −20° C.The reaction mixture was treated with 1-chloro-2-iodoethane (0.126 mL,1.71 mmol) and stirred for 15 min at 25° C. The reaction was dilutedwith H₂O (30 mL), extracted with Et₂O (3×20 mL), and the combinedorganic extracts were washed with saturated aqueous NaCl, dried (Na₂SO₄)and concentrated under reduced pressure. Flash chromatography (SiO₂,2.5×10 cm, 0-10% EtOAc/hexane gradient) yielded recovered 13 (51.8 mg,41%) and 14 (85.5 mg, 46%) as a white solid: mp 82-84° C.; ¹H NMR(CDCl₃, 400 MHz) δ 7.72 (d, J=8.2 Hz, 1H), 7.21 (t, J=6.0 Hz, 1H), 7.01(br s, 1H), 6.72 (dd, J=1.2, 8.2 Hz, 1H), 5.21 (s, 2H), 3.47 (s, 3H),1.49 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ 156.0, 152.5, 140.1, 129.5,113.4, 108.9, 94.8, 82.4, 80.9, 54.4, 28.3; IR (film) ν_(max) 3388,2977, 1736, 1592, 1515, 1465, 1406, 1252, 1226, 1153, 1005 cm⁻¹; FABHRMS(NBA/CsI) m/z 511.9351 (C₁₃H₁₈INO₄+C⁺ requires 511.9335). Anal. Calcdfor C₁₃H_(18INO) ₄: C, 41.18; H, 4.78; N, 3.69. Found: C, 41.19; H,5.11; N, 3.79.

[N-(tert-Butyloxycarbonyl)-N-(2-propen-1-yl)amino]-2-iodo-3-(methoxymethoxy)benzene(15): A solution of 14 (141 mg, 0.37 mmol) in 12 mL anhydrous DMF wascooled to −10° C., and treated with NaH (22.3 mg, 0.55 mmol) in smallportions. The resulting suspension was stirred for 15 min and treatedwith neat allyl bromide (0.16 mL, 1.56 mmol) in a slow dropwise manner.The reaction mixture was warmed to 25° C. and stirred for 1 h. Thereaction mixture was quenched with the addition of 5% aqueous NaHCO₃ (20mL), and the aqueous layer was extracted with EtOAc (3×10 mL). Thecombined organic extracts were washed with H₂O (5×10 mL), dried(Na₂SO₄), and condensed under reduced pressure to yield 15 as a 2:1mixture of amide rotamers as a yellow oil. Flash chromatography (SiO₂,2.5×10 cm, 10% EtOAc/hexane) yielded 15 (149 mg, 96%) as a colorlessoil: ¹H NMR (CDCl₃, 250 MHz) δ 7.17 (m, 1H), 6.95-6.78 (m, 2H),5.98-5.84 (m, 1H), 5.21 (s, 2H), 5.09-5.03 (m, 2H), 4.46 (m, 1H), 3.50(s, 3H), 1.50 and 1.31 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ 157.5 and157.0, 153.7, 146.1 and 145.7, 133.8 and 133.6, 129.5 and 129.0, 123.6and 123.5, 117.6 and 117.1, 113.5 and 113.2, 95.0 and 94.1, 80.5 and80.1, 56.4, 53.1, 51.9, 28.3 and 28.1; IR (film) ν_(max) 2975, 1688,1582, 1463, 1381, 1253, 1154, 1065, 990 cm⁻¹; FABHRMS (NBA/NaI) m/z420.0663 (C₁₆H₂₂INO₄+H⁺ requires 420.0672).

1-(tert-Butyloxycarbonyl)-4-(methoxymethoxy)-3-[[(2′,2′,6′,6′-tetramethylpiperidino)oxy]methyl]-2,3-dihydroindole(16): A solution of 15 (142 mg, 0.33 mmol) and TEMPO (160 mg, 1.0 mmol)in 14.3 mL anhydrous benzene was treated with Bu₃SnH (96 μL, 0.35 mmol).The solution was warmed at 50° C. and an additional 1.05 equiv of Bu₃SnH(96 μL, 0.35 mmol) was added twice during the next 30 min. Another 3.0equiv of TEMPO (160 mg, 1.0 mmol) was added in 3 mL anhydrous benzene,and an additional 1.05 equiv of Bu₃SnH added twice during the next 45min. After 1.5 h total the solution was cooled to 25° C., and thevolatiles were removed under reduced pressure. Flash chromatography(SiO₂, 2.5×10 cm, 0-8% EtOAc/hexane gradient) provided 16 (138 mg, 91%)as a yellow oil: ¹H NMR (CDCl₃, 250 MHz) δ 7.51 (br s, 1H), 7.11 (m,1H), 6.67 (d, J=8.7 Hz, 1H), 5.16 (s, 2H, 4.13 (dd, J=3.3, 11.4 Hz, 1H),4.07 (m, 1H), 3.93 (n, 1H), 3.76 (m, 1H), 3.54 (m, 1H), 3.46 (s, 3H),1.55 (s, 9H), 1.47-0.76 (m, 18H); ¹³C NMR (C₆D₆, 100 MHz) δ 154.2,152.2, 145.4, 129.8, 119.0, 109.6, 108.1, 94.4, 79.8, 77.3, 60.0, 55.7,52.4, 39.9, 33.3, 28.3, 20.1, 17.4; IR (film) ν_(max) 2974, 2931, 1707,1609, 1462, 1389, 1250, 1154, 1009 cm⁻¹; FABHRMS (NBA/CsI) m/z 581.1977(C₂₅H₄₀N₂O₅+H⁺ requires 581.1992).

1-(tert-Butyloxycarbonyl)-3-(hydroxymethyl)-4-(metboxymethoxy)-2,3-dihydroindole(17): A solution of 16 (135 mg, 0.30 mmol) in 10 mL 3:1:1 HOAc/H₂O/THFwas treated with Zn powder (780 mg, 12.0 mmol) and the resultingsuspension was warmed at 70° C. under a reflux condenser and withvigorous stirring for 2 h. The reaction mixture was cooled to 25° C.,and the Zn was removed by filtration through Celite (CH₂Cl₂ wash). Thevolatiles were removed under reduced pressure, and the resulting residuewas dissolved in 15 mL EtOAc and filtered. The solution was concentratedunder reduced pressure and subjected to flash chromatography (SiO₂,2.5×10 cm, 30-40% EtOAc/hexane gradient) to provide 16 (84 mg, 87%) as acolorless oil: ¹H NMR (CDCl₃, 400 MHz) δ 7.53 (br s, 1H), 7.11 (m, 1H),6.67 (d, J=8.8 Hz, 1H), 5.18 (d, J=8.6 Hz, 1H), 5.16 (d, J=8.6 Hz, 1H),4.02 (dd, J=10.1, 11.5 Hz, 1H), 3.84 (m, 2H), 3.71 (dd, J=5.9, 10.4 Hz,1H), 3.61 (m, 1), 3.46 (s, 3H), 1.53 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ153.6, 152.3, 129.7, 110.9, 110.0, 109.0, 107.8, 106.6, 94.2, 64.7,56.2, 51.3, 41.0, 28.3; IR (film) ν_(max) 3444, 2975, 1704, 1608, 1463,1392, 1252, 1153, 1004 cm⁻¹; FABHRMS (NBA/CsI) m/z 442.0642(C₁₆H₂₃NO₅+Cs⁺ requires 442.0631).

1-(tert-Butyloxycarbonyl)-4-(methoxymethoxy)-3-[[(methanesulfonyl)oxy]methyl]-2,3-dihydroindole(18): A solution of 17 (80 mg, 0.26 mmol) in 5 mL anhydrous CH₂Cl₂ wascooled to 0° C. and treated with Et₃N (79 μL, 0.57 mmol). After 10 min,MsCl (40 μL, 0.52 mmol) was added and the reaction mixture wassubsequently warmed to 25° C. and stirred for 3 h. The reaction solutionwas concentrated under reduced pressure. Flash chromatography (SiO₂,2.5×10 cm, 30% EtOAc/hexane) yielded pure 18 (94 mg, 94%) as a colorlessoil: ¹H NMR (CDCl₃, 250 MHz) δ 7.49 (m, 1H), 7.15 (m, 1H), 6.68 (d,J=9.0 Hz, 1H), 5.19 (d, J=8.8 Hz, 1H), 5.17 (d, J=8.8 Hz, 1H), 4.58 (dd,J=3.6, 9.7 Hz, 1H), 4.21 (app t, J=8.9 Hz, 1H), 4.00 (m, 2H), 3.79 (m,1H), 3.46 (s, 3H), 2,71 (s, 3H), 1.54 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz)δ 153.9, 152.1, 130.5, 110.2, 108.8, 107.7, 103.9, 94.4, 81.0, 69.9,56.2, 51.1, 37.9, 37.2, 28.2; IR (film) ν_(max) 2976, 1703, 1610, 1479,1463, 1391, 1355, 1254, 1175, 1154, 1061, 952 cm⁻; FABHRMS (NBA/CsI) m/z520.0388 (C₁₇H₂₅NO₇S+Cs⁺ requires 520.0406).

DNA Alkylation Studies: Selectivity and Efficiency: Eppendorf tubescontaining singly ³²p 5′-end-labeled double-stranded DNA (9 μL) (Boger,D. L., et al., Tetrahedron 1991, 47, 2661.) in TE buffer (10 mM Tris, 1mM EDTA, pH 7.5) were treated with the agents in DMSO (1 μL, at thespecified concentrations). The solutions were mixed by vortexing andbrief centrifugation and subsequently incubated at 4° C., 25° C. or 37°C. for 24-72 h. The covalently modified DNA was separated from unboundagent by EtOH precipitation of the DNA The EtOH precipitations werecarried out by adding t-RNA as a carrier (1 μL, 10 μg/μL), 3 M NaOAc(0.1 volume) and −20° C. EtOH (2.5 volumes). The solutions were mixedand chilled at −78° C. in a REVCO freezer for 1 h or longer. The DNA wasreduced to a pellet by centrifugation at 4° C. for 15 min and washedwith −20° C. 70% EtOH (in TE containing 0.2 M NaCl). The pellets weredried in a Savant Speed Vac concentrator and resuspended in TE buffer(10 μL). The solutions of alkylated DNA were warmed at 100° C. for 30min to induce cleavage at the adenine N3 alkylation sites. After briefcentrifugation, formamide dye solution (5 μL) was added. Prior toelectrophoresis, the samples were denatured by warming at 100° C. for 5min, placed in an ice bath, centrifuged briefly, and the supplement (2.8μL) was loaded onto a gel. Sanger dideoxynucleotide sequencing reactionswere run as standards adjacent to the agent treated DNA reactionsamples. Polyacrylamide gel electrophoresis (PAGE) was run on a 8%sequencing gel under denaturing conditions (19:1 acrylamide:N,N-methylenebisacrylamide, 8 M urea) in TBE buffer (100 mM Tris, 100 mMboric acid, 0.2 mM Na₂EDTA). PAGE was pre-run for 30 min with formamidedye solution prior to loading the samples. autoradiography of dried gelswas carried out at −78° C. using kodak O-omat AR film and pickerspectra™ intensifying screen.

What is claimed:
 1. A process for synthesizing a third chemicalintermediate of iso-CI analogs, the process comprising the followingsteps: Step A: converting a first intermediate to a second intermediateas follows:

 and then Step B: converting the second intermediate of said Step A tothe third intermediate as follows:


2. A process for synthesizing a third chemical intermediate of iso-CIanalogs, the process comprising the following steps: Step A. convertinga first intermediate to a second intermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe third intermediate as follows:


3. A process for synthesizing a third chemical intermediate of iso-CIanalogs, the process comprising the following steps: Step A: convertinga first intermediate to a second intermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe third intermediate as follows:


4. A process for synthesizing a third chemical intermediate of iso-CIanalogs, the process comprising the following steps: Step A: convertinga first intermediate to a second intermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe third intermediate as follows:


5. A process for synthesizing a third chemical intermediate of iso-CIanalogs, the process comprising the following steps: Step A: convertinga first intermediate to a second intermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe third intermediate as follows:

wherein R is a radical selected from the following group:


6. A process for synthesizing an iso-CI analog, the process comprisingthe following steps: Step A: converting a first intermediate to a secondintermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe iso-CI analog as follows:

wherein R is a radical selected from the following group:


7. A process for synthesizing a third chemical intermediate of iso-CBIanalogs, the process comprising the following steps: Step A: convertinga first intermediate to a second intermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe third intermediate as follows:


8. A process for synthesizing a third chemical intermediate of iso-CBIanalogs, the process comprising the following steps: Step A: convertinga first intermediate to a second intermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe third intermediate as follows:


9. A process for synthesizing a third chemical intermediate of iso-CBIanalogs, the process comprising the following steps: Step A: convertinga first intermediate to a second intermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe third intermediate as follows:


10. A process for synthesizing a third chemical intermediate of iso-CBIanalogs, the process comprising the following steps: Step A: convertinga first intermediate to a second intermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe third intermediate as follows:


11. A process for synthesizing a third chemical intermediate of iso-CBIanalogs, the process comprising the following steps: Step A: convertinga first intermediate to a second intermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe third intermediate as follows:


12. A process for synthesizing a third chemical intermediate of iso-CBIanalogs, the process comprising the following steps: Step A: convertinga first intermediate to a second intermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe third intermediate as follows:

 wherein R is a radical selected from the following group:


13. A process for synthesizing an iso-CBI analog, the process comprisingthe following steps: Step A: converting a first intermediate to a secondintermediate as follows:

 and then Step B: converting the second intermediate of said Step A tothe iso-CBI analog as follows:

wherein R is a radical selected from the following group: